CRISPR-TALENs
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Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease

Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease | CRISPR-TALENs | Scoop.it

Gratz et al., Genetics

 

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.

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Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System | CRISPR-TALENs | Scoop.it
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A CRISPR method for genome-wide screening : Nature Genetics : Nature Publishing Group

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Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation | CRISPR-TALENs | Scoop.it
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CRISPR-Cas systems for editing, regulating and targeting genomes : Nature Biotechnology : Nature Publishing Group

CRISPR-Cas systems for editing, regulating and targeting genomes : Nature Biotechnology : Nature Publishing Group | CRISPR-TALENs | Scoop.it
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CRISPR RNA–guided activation of endogenous human genes

CRISPR RNA–guided activation of endogenous human genes | CRISPR-TALENs | Scoop.it

Morgan et al, 2013, Nat Met

Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.

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Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems

Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems | CRISPR-TALENs | Scoop.it

Li et al., 2013, Nat Biotech

 

CRISPRs are clustered, regularly interspaced, short palindromic repeats present in many bacteria and archaea genomes. Proteins encoded by CRISPR-associated (Cas) genes serve as guardians of the genome, which target foreign DNA at specific sites by means of small CRISPR RNA (crRNA)-guided DNA recognition and degradation

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Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9

Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 | CRISPR-TALENs | Scoop.it

Li et al., 2013, Nat Biotech

 

Elucidation and manipulation of human, animal and plant genomes is key to basic biology research, medical advances and crop improvement. The development of targeted genome editing, particularly homologous recombination–based gene replacement, is of great value in all organisms. Recent advances in engineered nucleases with programmable DNA-binding specificities, such…

  

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Clem Stanyon's curator insight, August 10, 2013 11:20 PM

Paper for genome editing of plants by the Cas/CRISPR system. Note that many crop plants are not merely diploid, like our own species and other metazoans, but tetrapolid or, in the case of wheat, hexaploid. This means that getting the target loci modified on all of the homologous chromosomes of the organism is a much bigger challenge. Thus, a very high efficiency of targeting is even more important for higher-ploidy organisms.

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Science: The CRISPR Craze (2013)

Science: The CRISPR Craze (2013) | CRISPR-TALENs | Scoop.it

Bacteria have a kind of adaptive immune system, which enables them to fight off repeated attacks by specific viruses, that works through precise targeting of DNA. In January, four research teams reported harnessing the system, called CRISPR, to target the destruction of specific genes in human cells. And in the following 8 months, various groups have used it to delete, add, activate or suppress targeted genes in human cells, mice, rats, zebrafish, bacteria, fruit flies, yeast, nematodes and crops, demonstrating broad utility for the technique. With CRISPR, scientists can create mouse models of human diseases much more quickly than before, study individual genes much faster, and easily change multiple genes in cells at once to study their interactions.

 


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High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells : Nature Biotechnology : Nature Publishing Group

CAS9 nucleases, a new tool for genome editing, show significant offtarget activity.
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Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease

Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease | CRISPR-TALENs | Scoop.it

NATURE BIOTECHNOLOGY | RESEARCH | BRIEF COMMUNICATIONS

 

We employ the CRISPR-Cas system of Streptococcus pyogenes as programmable RNA-guided endonucleases (RGENs) to cleave DNA in a targeted manner for genome editing in human cells. We show that complexes of the Cas9 protein and artificial chimeric RNAs efficiently cleave two genomic sites and induce indels with frequencies of up to 33%.

 

 

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Serotonin Receptors Expressed in Drosophila Mushroom Bodies Differentially Modulate Larval Locomotion

Serotonin Receptors Expressed in Drosophila Mushroom Bodies Differentially Modulate Larval Locomotion | CRISPR-TALENs | 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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Improving CRISPR-Cas nuclease specificity using truncated guide RNAs : Nature Biotechnology : Nature Publishing Group

Improving CRISPR-Cas nuclease specificity using truncated guide RNAs : Nature Biotechnology : Nature Publishing Group | CRISPR-TALENs | Scoop.it
Guide RNAs with shorter regions of complementarity to target sites reduce off-target effects.
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Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila

Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila | CRISPR-TALENs | Scoop.it
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Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library : Nature Biotechnology : Nature Publishing Group

Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library : Nature Biotechnology : Nature Publishing Group | CRISPR-TALENs | Scoop.it
CRISPR-Cas9 genome editing technology is used for genome-wide genetic screens in mouse embryonic stem cells.
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Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9

Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9 | CRISPR-TALENs | 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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Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects : Nature Methods : Nature Publishing Group

Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects : Nature Methods : Nature Publishing Group | CRISPR-TALENs | Scoop.it
This paper describes the use of paired Cas9 nickases to edit the mammalian genome with no detectable off-target effects.
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RNA-guided gene activation by CRISPR-Cas9–based transcription factors

RNA-guided gene activation by CRISPR-Cas9–based transcription factors | CRISPR-TALENs | Scoop.it

Pablo et al, 2013, Nat Met

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

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Heritable gene targeting in the mouse and rat using a CRISPR-Cas system

Heritable gene targeting in the mouse and rat using a CRISPR-Cas system | CRISPR-TALENs | Scoop.it

Li et al., 2013, Nat Biotech

 

CRISPR-Cas systems have been developed as an efficient gene editing technology in cells and model organisms. Here we use a CRISPR-Cas system to induce genomic DNA fragment deletion in mice by co-injecting two single-guide RNAs (sgRNAs) targeting the Uhrf2 locus with Cas9 mRNA. Furthermore, we report the generation…

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Targeted genome modification of crop plants using a CRISPR-Cas system

Targeted genome modification of crop plants using a CRISPR-Cas system | CRISPR-TALENs | Scoop.it

Shan et al., 2013, Nat Biotech

 

Although genome editing technologies using zinc finger nucleases (ZFNs)1 and transcription activator-like effector nucleases (TALENs)2 can generate genome modifications, new technologies that are robust, affordable and easy to engineer are needed. Recent advances in the study of the prokaryotic adaptive immune system, involving type II clustered, regularly interspaced

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DNA targeting specificity of RNA-guided Cas9 nucleases

DNA targeting specificity of RNA-guided Cas9 nucleases | CRISPR-TALENs | Scoop.it

Via Gerd Moe-Behrens
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Gerd Moe-Behrens's curator insight, July 23, 2013 4:09 PM


Improved technique makes it easier to add or delete genes in living cells, with less risk of off-target DNA damage. (comment see below)

*DNA targeting specificity of RNA-guided Cas9 nucleases*

by
Patrick D Hsu,David A Scott,Joshua A Weinstein,F Ann Ran,Silvana Konermann,Vineeta Agarwala,Yinqing Li,Eli J Fine,Xuebing Wu,Ophir Shalem,Thomas J Cradick,Luciano A Marraffini,Gang Bao& Feng Zhang

"The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses."

 http://bit.ly/15e3QI1

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*Research update: Genome editing becomes more accurate*

by 
Anne Trafton

"Earlier this year, MIT researchers developed a way to easily and efficiently edit the genomes of living cells. Now, the researchers have discovered key factors that influence the accuracy of the system, an important step toward making it safer for potential use in humans, says Feng Zhang, leader of the research team. 

 With this technology, scientists can deliver or disrupt multiple genes at once, raising the possibility of treating human disease by targeting malfunctioning genes. To help with that process, Zhang’s team, led by graduate students Patrick Hsu and David Scott, has now created a computer model that can identify the best genetic sequences to target a given gene. “Using this, you will be able to identify ways to target almost every gene. Within every gene, there are hundreds of locations that can be edited, and this will help researchers narrow down which ones are better than others,” says Zhang, the W.M. Keck Assistant Professor in Biomedical Engineering at MIT and senior author of a paper describing the new model, appearing in the July 21 online edition of Nature Biotechnology. The genome-editing system, known as CRISPR, exploits a protein-RNA complex that bacteria use to defend themselves from infection. The complex includes short RNA sequences bound to an enzyme called Cas9, which slices DNA. These RNA 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.  This technique offers a much faster and more efficient way to create transgenic mice, which are often used to study human disease. Current methods for creating such mice require adding small pieces of DNA to mouse embryonic cells. However, the process is inefficient and time-consuming.  With CRISPR, many genes are edited at once, and the entire process can be done in three weeks, says Zhang, who is a core member of the Broad Institute and MIT’s McGovern Institute for Brain Research. The system can also be used to create genetically modified cell lines for lab experiments much more efficiently. Fine-tuning Since Zhang and his colleagues first described the original system in January, more than 2,000 labs around the world have started using the system to generate their own genetically modified cell lines or animals. In the new paper, the researchers describe improvements in both the efficiency and accuracy of gene editing.  To modify genes using this system, an RNA “guide strand” complementary to a 20-base-pair sequence of targeted DNA is delivered to cells. After the RNA strand binds to the target DNA, it recruits the Cas9 enzyme, which snips the DNA in the correct location. The researchers discovered they could minimize the chances of the Cas9-RNA complex accidentally cleaving the wrong site by making sure the target sequence is not too similar to other sequences found in the genome. They found that if an off-target sequence differs from the target sequence by three or fewer base pairs, the editing complex will likely also cleave that sequence, which could have deleterious effects for the cell.  The team’s new computer model can search any sequence within the mouse or human genome and identify 20-base-pair sequences within that region that have the least overlap with sequences elsewhere in the genome.  Another way to improve targeting specificity is by adjusting the dosage of the guide RNA, the researchers found. In general, decreasing the amount of RNA delivered minimizes damage to off-target sites but has a much smaller effect on cleavage of the target sequence. For each sequence, the “sweet spot” with the best balance of high on-target effects and low off-target effects can be calculated, Zhang says. “The real value of this paper is that it does a very comprehensive and systematic analysis to understand the causes of off-target effects. That analysis suggests a lot of possible ways to eliminate or reduce off-target effects,” says Michael Terns, a professor of biochemistry and molecular biology at the University of Georgia who was not part of the research team. Zhang and his colleagues also optimized the structure of the RNA guide needed for efficient activation of Cas9. In the January paper describing the original system, the researchers found that two separate RNA strands working together — one that binds to the target DNA and another that recruits Cas9 — produced better results than when those two strands were fused together before delivery. However, in experiments reported in the new paper, the researchers found that they could boost the efficiency of the fused RNA strand by making the strand longer. These longer RNA guide strands include a hairpin structure that may stabilize the molecules and help them interact with Cas9, Zhang says. Zhang’s team is now working on further improving the specificity of the system, and plans to start generating cell lines and animals that could be used to study how the brain develops and builds neural circuits. By disrupting genes known to be involved in those processes, they can learn more about how they work and how they are impaired in neurological disease. "


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An efficient strategy for TALEN-mediated genome engineering in Drosophila

An efficient strategy for TALEN-mediated genome engineering in Drosophila | CRISPR-TALENs | 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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DNA targeting specificity of RNA-guided Cas9 nucleases : Nature Biotechnology : Nature Publishing Group

DNA targeting specificity of RNA-guided Cas9 nucleases : Nature Biotechnology : Nature Publishing Group | CRISPR-TALENs | Scoop.it
Analyses of the determinants of the specificity of Cas9 nuclease provide rules for selecting optimal target sites.
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TALE-mediated modulation of transcriptional enhancers in vivo - Nature Methods

TALE-mediated modulation of transcriptional enhancers in vivo - Nature Methods | CRISPR-TALENs | Scoop.it

Crocker & Stern 2013

We tested whether transcription activator–like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.


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