Biotechnologies
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CRISPR-CAS9 Reverses Disease Symptoms in Living Animals for First Time

CRISPR-CAS9 Reverses Disease Symptoms in Living Animals for First Time | Biotechnologies | Scoop.it

MIT scientists report the use of a CRISPR methodology to cure mice of a rare liver disorder caused by a single genetic mutation. They say their study (“Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype”), published in Nature Biotechnology, offers the first evidence that this gene-editing technique can reverse disease symptoms in living animals. CRISPR, which provides a way to snip out mutated DNA and replace it with the correct sequence, holds potential for treating many genetic disorders, according to the research team.

 

“What's exciting about this approach is that we can actually correct a defective gene in a living adult animal,” says Daniel Anderson, Ph.D., the Samuel A. Goldblith associate professor of chemical engineering at MIT, a member of the Koch Institute for Integrative Cancer Research, and the senior author of the paper.

 

The recently developed CRISPR system relies on cellular machinery that bacteria use to defend themselves from viral infection. Researchers have copied this cellular system to create gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, telling Cas9 where to make its cut.

 

At the same time, the researchers also deliver a DNA template strand. When the cell repairs the damage produced by Cas9, it copies from the template, introducing new genetic material into the genome. Scientists envision that this kind of genome editing could one day help treat diseases such as hemophilia, and others that are caused by single mutations.

 

For this study, the researchers designed three guide RNA strands that target different DNA sequences near the mutation that causes type I tyrosinemia, in a gene that codes for an enzyme called FAH. Patients with this disease, which affects about 1 in 100,000 people, cannot break down the amino acid tyrosine, which accumulates and can lead to liver failure. Current treatments include a low-protein diet and a drug called NTCB, which disrupts tyrosine production.

 

In experiments with adult mice carrying the mutated form of the FAH enzyme, the researchers delivered RNA guide strands along with the gene for Cas9 and a 199-nucleotide DNA template that includes the correct sequence of the mutated FAH gene.

 

“Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ~1/250 liver cells,” wrote the investigators. “Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype.”

 

While the team used a high pressure injection to deliver the CRISPR components, Dr. Anderson envisions that better delivery approaches are possible. His lab is now working on methods that may be safer and more efficient, including targeted nanoparticles. 


Via Dr. Stefan Gruenwald
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Tomatoes: The world's favorite fruit, only better-tasting and longer-lasting

Tomatoes: The world's favorite fruit, only better-tasting and longer-lasting | Biotechnologies | Scoop.it
Research with GM purple tomatoes could lead to improved varieties of tomatoes with consumer and commercial benefits through conventional breeding or GM. The findings could also be applied to other soft fruit such as strawberries.
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En Israël, des arbres OGM pour remplacer les énergies fossiles ?

En Israël, des arbres OGM pour remplacer les énergies fossiles ? | Biotechnologies | Scoop.it
L'introduction d'un gène capable de booster de 40 % la croissance de peupliers et des eucalyptus pourrait permettre de nouveaux débouchés dans l'industrie des biocarburants et celle du bois.
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'Growing' medicines in plants requires new regulations, experts say

'Growing' medicines in plants requires new regulations, experts say | Biotechnologies | Scoop.it
Scientists say amending an EU directive on GMOs could help stimulate innovation in making vaccines, cheaper pharmaceuticals and organic plastics using plants.
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CliniGene - Gene Therapy European Network - Gene Therapy

CliniGene - Gene Therapy European Network - Gene Therapy | Biotechnologies | Scoop.it
European Network for the advancement of Clinical Gene Transfer & Therapy : EC-NoE fostering interaction of all stakeholders in the field in order to facilitate and help harmonise Ethical, Quality, Efficacy and Regulatory Issues.
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New genes for short-sightedness: 24 new genes that cause refractive errors and myopia identified

Myopia is a major cause of blindness and visual impairment worldwide, and currently there is no cure. New findings reveal genetic causes of the trait, which could lead to finding better treatments or ways of preventing the condition in the future.
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Device made of DNA inserted into bacterial cell works like a diagnostic computer

Device made of DNA inserted into bacterial cell works like a diagnostic computer | Biotechnologies | Scoop.it
A biological device made of DNA inserted into a bacterial cell works like a tiny diagnostic computer.
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Biologists engineer algae to make complex anti-cancer 'designer' drug

Biologists engineer algae to make complex anti-cancer 'designer' drug | Biotechnologies | Scoop.it
Biologists have succeeded in genetically engineering algae to produce a complex and expensive human therapeutic drug used to treat cancer.
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Actu santé : CRISE CARDIAQUE: Thérapie génique et facteur de croissance peuvent réparer le cœur

réseau de professionnels de santé
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Artificial pancreas: The way of the future for treating type 1 diabetes

Artificial pancreas: The way of the future for treating type 1 diabetes | Biotechnologies | Scoop.it
Researchers have conducted a trial comparing a dual-hormone artificial pancreas with conventional diabetes treatment using an insulin pump and showed improved glucose levels and lower risks of hypoglycemia.
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UK scientists bid to mimic plant energy creation

UK scientists bid to mimic plant energy creation | Biotechnologies | Scoop.it
£800,000 research project will attempt to replicate photosynthesis in order to make a more efficient renewable fuel

Via Rob Dawson
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Après un an d’arrêt, la recherche sur les virus mutants de la grippe aviaire reprend

Après un an d’arrêt, la recherche sur les virus mutants de la grippe aviaire reprend | Biotechnologies | Scoop.it
Après un an d’arrêt, la recherche sur les virus mutants de la grippe aviaire reprend
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Truncated guide RNAs drastically improve specificity of CRISPR-Cas nucleases

Truncated guide RNAs drastically improve specificity of CRISPR-Cas nucleases | Biotechnologies | Scoop.it
A simple adjustment to a powerful gene-editing tool may be able to improve its specificity. Investigators have found that adjusting the length of the the guide RNA component of the synthetic enzymes called CRISPR-Cas RNA-guided nucleases can substantially reduce the occurrence of off-target DNA mutations.

 

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools1, 2, 3. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target4, 5, 6. A research team recently reports that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). These results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.


"Simply by shortening the length of the gRNA targeting region, we saw reductions in the frequencies of unwanted mutations at all of the previously known off-target sites we examined," says J. Keith Joung, MD, PhD, associate chief for Research in the MGH Department of Pathology and senior author of the report. "Some sites showed decreases in mutation frequency of 5,000-fold or more, compared with full length gRNAs, and importantly these truncated gRNAs -- which we call tru-gRNAs -- are just as efficient as full-length gRNAs at reaching their intended target DNA segments."

 

CRISPR-Cas RGNs combine a gene-cutting enzyme called Cas9 with a short RNA segment and are used to induce breaks in a complementary DNA segment in order to introduce genetic changes. Last year Joung's team reported finding that, in human cells, CRISPR-Cas RGNs could also cause mutations in DNA sequences with differences of up to five nucleotides from the target, which could seriously limit the proteins' clinical usefulness. The team followed up those findings by investigating a hypothesis that could seem counterintuitive, that shortening the gRNA segment might reduce off-target mutations.


"Some of our experiments from last year suggested that one could mismatch a few nucleotides at one end of the gRNA complementarity region without affecting the targeting activity," Joung explains. "That led us to wonder whether removing these nucleotides could make the system more sensitive to mismatches in the remaining sequence."

 

Based on a natural system a species of bacteria uses against other pathogens, the CRISPR-Cas RGNs most widely used by researchers includes a 20-nucleotide targeting region within the gRNA. To test their theory, the MGH team constructed RGNs with progressively shorter gRNAs and found that, while gRNAs with targeting segments of 17 or 18 nucleotides were as or more efficient than full-length gRNAs in reaching their targets, those with 15- or 16-nucleotide targeting segments had reduced or no targeting activity. Subsequent experiments found that 17-nucleotide truncated RGNs efficiently induced the desired mutations in human cells with greatly reduced or undetectable off-target effects, even at sites with only one or two mismatches.

 

"While we don't fully understand the mechanism by which tru-gRNAs reduce off-target effects, our hypothesis is that the original system might have more energy than it needs, enabling it to cleave even imperfectly matched sites," says Joung, who is an associate professor of Pathology at Harvard Medical School. "By shortening the gRNA, we may reduce the energy to a level just sufficient for on-target activity, making the nuclease less able to cleave off-target sites. But more work is needed to define exactly why tru-gRNAs have reduced off-target effects."



Via Dr. Stefan Gruenwald
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Engineering algae to make the 'wonder material' nanocellulose for biofuels and more

Engineering algae to make the 'wonder material' nanocellulose for biofuels and more | Biotechnologies | Scoop.it
Genes from the family of bacteria that produce vinegar, Kombucha tea and nata de coco have become stars in a project -- which scientists today said has reached an advanced stage -- that would turn algae into solar-powered factories for producing...
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Designer TAL effectors induce disease susceptibility and resistance to Xanthomonas oryzae pv. oryzae in rice

Designer TAL effectors induce disease susceptibility and resistance to Xanthomonas oryzae pv. oryzae in rice | Biotechnologies | Scoop.it

TAL (transcription activator-like) effectors from Xanthomonas bacteria activate the cognate host genes, leading to disease susceptibility or resistance dependent on the genetic context of host target genes. The modular nature and DNA recognition code of TAL effectors enable custom-engineering of designer TAL effectors (dTALE) for gene activation. However, the feasibility of dTALEs as transcription activators for gene functional analysis has not been demonstrated. Here we report the use of dTALEs, as expressed and delivered by the pathogenic Xanthomonas oryzae pv. oryzae (Xoo), in revealing the new function of two previously identified disease-related genes and the potential of one developmental gene for disease susceptibility in rice/Xoo interactions. The dTALE gene dTALE-xa27, designed to target the susceptible allele of the resistance gene Xa27, elicited a resistant reaction in the otherwise susceptible rice cultivar IR24. Four dTALE genes were made to induce the four annotated Xa27 homologous genes in rice cultivar Nipponbare, but none of the four induced Xa27-like genes conferred resistance to the dTALE-containing Xoo strains. A dTALE gene was also generated to activate the recessive resistance gene xa13, an allele of the disease susceptibility gene Os8N3 (also named Xa13 or OsSWEET11, a member of sucrose efflux transporter SWEET gene family). The induction of xa13 by the dTALE rendered the resistant rice IRBB13 (xa13/xa13) susceptible to Xoo. Finally, OsSWEET12, an as yet uncharacterized SWEET gene with no corresponding naturally occurring TAL effector identified, conferred susceptibility to the Xoo strains expressing the corresponding dTALE genes. Our results demonstrate that dTALEs can be delivered through the bacterial secretion system to activate genes of interest for functional analysis in plant.


Via Ricardo Oliva, dromius
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Synthetic circuit allows dialing gene expression up or down in human cells

Synthetic circuit allows dialing gene expression up or down in human cells | Biotechnologies | Scoop.it
Scientists who built a synthetic gene circuit that allowed for the precise tuning of a gene's expression in yeast have now refined this new research tool to work in human cells, according to new research.
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Toward an artificial pancreas | Machines Like Us

Toward an artificial pancreas | Machines Like Us | Biotechnologies | Scoop.it
Researchers take an important step toward making the artificial pancreas a reality.

IRCM researchers, led by endocrinologist Dr.
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Rapid and efficient gene modification in rice and Brachypodium using TALENs - Mol. Plant

Rapid and efficient gene modification in rice and Brachypodium using TALENs - Mol. Plant | Biotechnologies | Scoop.it

Four rice genes and eight Brachypodium genes were targeted to generate knockout mutations. A protoplast transient assay was developed in both rice and Brachypodium. TALEN-encoding constructs were introduced into protoplasts using polyethylene glycol (PEG). Seven out of eleven TALENs showed mutagenesis frequencies ranging from
4% to 14%. The TALENs targeting the 4 rice and 8 Brachypodium genes were transformed into plants. The constructs were introduced into embryonic cells of rice or Brachypodium using Agrobacterium tumefaciens.

 

Two rice TALENs separated by 1322 bp in the OsBADH2
gene, were co-introduced into rice protoplasts. These two TALEN pairs were next co-delivered into rice calli by particle bombardment. Ninety-eight hygromycin-resistant calli were screened after 6 weeks of selection, and three types of modifications were identified — simple modifications (small deletions or insertions) at either target site, large deletions of the sequence between the
two target sites and inversion of the sequence between the two target sites.

These results support the idea that TALEN-induced genomic deletions were mediated via NHEJ. Targeted deletions would make possible the selective removal of gene clusters and enables scientists to delete intergenic regions, introns, regulatory elements and noncoding RNAs.
To our knowledge, this is the first study demonstrating highly efficient targeted
knockouts in multiple genes in the model monocot species Brachypodium. We anticipate TALEN technology will make targeted gene modification a routine practice not just for these model monocots but also for economically important crops, such as maize and wheat.


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Jennifer Mach's curator insight, January 7, 2013 5:48 PM

In case you missed this one (I did), to save you looking it up on Wikipedia, "Transcription Activator-Like Effector Nucleases (TALENs) are artificial restriction enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain."

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Giant tobacco plants that stay young forever

Giant tobacco plants that stay young forever | Biotechnologies | Scoop.it
Tobacco plants bloom when they are just a few months old -- and then they die. Now, researchers have located a genetic switch which can keep the plants young for years and which permits unbounded growth.
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Et les sourds entendront… - leJDD.fr

Et les sourds entendront… - leJDD.fr | Biotechnologies | Scoop.it
Les thérapies génique et cellulaire sont les deux voies de recherche pour traiter la surdité d’ici à quinze ans. Il faudra dans les deux cas injecter dans la cochlée, soit une préparation virale (t...
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Safer way to vaccinate? Polymer film that gradually releases DNA coding for viral proteins may beat traditional vaccines

Safer way to vaccinate? Polymer film that gradually releases DNA coding for viral proteins may beat traditional vaccines | Biotechnologies | Scoop.it
Researchers have described a new type of vaccine-delivery film that holds promise for improving the effectiveness of DNA vaccines.
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Biotechnologie - L'ADN sera-t-il le disque dur du futur?

Biotechnologie - L'ADN sera-t-il le disque dur du futur? | Biotechnologies | Scoop.it
Des chercheurs viennent de faire la démonstration de l'étonnante capacité de l'ADN synthétique à stocker des textes, des images ou des sons.
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Skin transformed into brain cells

Skin transformed into brain cells | Biotechnologies | Scoop.it
Skin cells have been converted directly into cells which develop into the main components of the brain, by researchers in California.
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