Genome Engineer
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iPS Engineering Hub™

iPS Engineering Hub™ | Genome Engineer | Scoop.it
From donor samples to engineered iPS cell lines in just 6 months!
The iPS Engineering Hub™ service provides you with customized iPS cell lines engineered by taking advantage of our TALEN™ technology.
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Rescooped by Jun Liu from CRISPR-Cas System for Eukaryotic Genome Engineering
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Addgene: Calarco Lab CRISPR Plasmids

Addgene: Calarco Lab CRISPR Plasmids | Genome Engineer | Scoop.it

Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA. NAT METHODS. 2013 Jun


Via Amir Taheri Ghahfarokhi
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CRISPR based genome editing - the future of molecular biology.

CRISPR based genome editing - the future of molecular biology. | Genome Engineer | Scoop.it
It isn't often that I make such seemingly outlandish claims in the title of a blog-post, but this particular technology, CRISPR-based genome editing, I believe deserves the hype. CRISPR stands for ...
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Multiplex genome engineering using CRISPR/Cas systems

Multiplex genome engineering using CRISPR/Cas systems | Genome Engineer | Scoop.it

Cong et al., 2013, Science

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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TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction

TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction | Genome Engineer | Scoop.it

Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0; https://boglab.plp.iastate.edu/) that enables design of custom TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest. Search parameters can be set by the user to work with any TAL effector or TAL effector nuclease architecture. Applications range from designing highly specific DNA targeting tools and identifying potential off-target sites to predicting effector targets important in plant disease.

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Mojo hand, a TALEN design tool for genome editing applications - BMC Bioinformatics

http://www.talendesign.org/mojohand_main.php

 

We present the web-based program Mojo Hand for designing TAL and TALEN constructs for genome editing applications (www.talendesign.org). We describe the algorithm and its implementation. The features of Mojo Hand include (1) automatic download of genomic data from the National Center for Biotechnology Information, (2) analysis of any DNA sequence to reveal pairs of binding sites based on a user-defined template, (3) selection of restriction enzyme recognition sites in the spacer between the TAL monomer binding sites including options for the selection of restriction enzyme suppliers, and (4) output files designed for subsequent TALEN construction using the Golden Gate assembly method.


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With 'gene-editing' scientists have produced a disease resistant piglet - MercoPress

With 'gene-editing' scientists have produced a disease resistant piglet - MercoPress | Genome Engineer | Scoop.it
With 'gene-editing' scientists have produced a disease resistant piglet MercoPress 'Gene-editing' involves researchers snipping the animal's DNA and inserting new genetic material, in effect changing a single one of the three billion 'letters' that...
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Targeted nucleases: spreading the joy : Nature Methods

Methods for targeted genome editing are developing at a fast pace. Are there barriers to their widespread use in research?

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Rescooped by Jun Liu from CRISPR-Cas System for Eukaryotic Genome Engineering
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Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish | Genome Engineer | Scoop.it

Xiao et al, 2013, NAR

 

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.


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Clem Stanyon's curator insight, June 17, 2013 5:03 AM

Not far from this paper to imagining that we could cause reversions of cancer-causing inversions...or putting together entirely novel genomes, also known as 'synthetic biology.'

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Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems | Genome Engineer | Scoop.it

The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency.

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The Buzz on the Cut: From Dream to Reality - Zone in With Zon

The Buzz on the Cut: From Dream to Reality - Zone in With Zon | Genome Engineer | Scoop.it
TALEN, CRISPR, zinc finger nuclease, gene therapy, cell therapy, mRNA, ZFN
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Genome editing in human pluripotent stem cells | StemBook

Genome editing in human pluripotent stem cells | StemBook | Genome Engineer | Scoop.it

Genome editing is used to make targeted modifications to the genome of eukaryotic cells. There are many potential applications of genome editing in human pluripotent stem cells (hPSCs) including the generation of knockout and reporter cell lines. This protocol describes a system for efficient genome editing in hPSCs using engineered transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat (CRISPR) technology.


Via Amir Taheri Ghahfarokhi
Jun Liu's insight:

TALEN libray!

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Cell Press Webinar Series: Genome editing of stem cells

Cell Press Webinar Series: Genome editing of stem cells | Genome Engineer | Scoop.it
interested in genome editing--watch this webinar to hear the latest http://t.co/FwzTcBbgXs via @CellCellPress
Jun Liu's insight:

Dr Feng Zhang (Broad and McGovern Institutes)talked about CRISPR.

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Enhanced Efficiency of Human Pluripotent Stem Cell Genome Editing through Replacing TALENs with CRISPRs

Enhanced Efficiency of Human Pluripotent Stem Cell Genome Editing through Replacing TALENs with CRISPRs | Genome Engineer | Scoop.it

Ding et al. Cell Stem Cells 2013

Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are new classes of genome-editing tools that target desired genomic sites in mammalian cells (Miller et al., 2011; Hockemeyer et al., 2011; Cong et al., 2013; Mali et al., 2013; Jinek et al., 2013). TALENs bind as a pair around a genomic site in which a double-strand break (DSB) is introduced by a dimer of FokI nuclease domains. Recently published type II CRISPR/Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes a 20-nucleotide DNA sequence (“protospacer”) beginning with G and immediately preceding an NGG motif recognized by Cas9—constituting a G(N)19NGG target DNA sequence—resulting in a DSB three nucleotides upstream of the NGG motif (Jinek et al., 2012). However it is generated, the DSB instigates either nonhomologous end-joining (NHEJ), which is error-prone and conducive to frameshift mutations (indels) that knock out gene alleles, or homology-directed repair (HDR), which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome.

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High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells | Genome Engineer | Scoop.it

Fu et al., 2013, Nat Biotech

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell–based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.


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Freddy Monteiro's curator insight, June 24, 2013 9:26 PM

Well, at least it is not the end. The system can stll be improved. What about other organisms? Do they also present a similar rate of non-specific mutagenesis?

Freddy Monteiro's comment, June 24, 2013 9:41 PM
Clem Stanyon has a particularly relevant comparison with zinc-finger endonuculeases in his scoopit post: http://sco.lt/5BJsTh
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TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA-targeting proteins - Trends Cell Biol

TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA-targeting proteins - Trends Cell Biol | Genome Engineer | Scoop.it

 

(via Tom Schreiber, thx)

Doyle et al, 2013

Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria of the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA-binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting.


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CRISPR/Cas System Genome Editing - EpiGenie

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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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 | Genome Engineer | 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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Genome editing of human pluripotent stem cells to generate human cellular disease models

Read new Review @DMM_Journal on genome editing of pluripotent #stemcells. Full text available for free! http://t.co/vjbufzJo4I #isscr
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