CRISPR-Cas System for Eukaryotic Genome Engineering
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Biotechnology: Rewriting a genome

Biotechnology: Rewriting a genome | CRISPR-Cas System for Eukaryotic Genome Engineering | 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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CRISPR-Cas System for Eukaryotic Genome Engineering
The ability to carry out multiplex genome editing in mammalian cells enables powerful applications across basic science, biotechnology, and medicine.
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In vivo genome editing using Staphylococcus aureus Cas9

In vivo genome editing using Staphylococcus aureus Cas9 | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

Ran et al. (2015)

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

Amir Taheri Ghahfarokhi's insight:

Hello My community :) After nearly two years after my latest scoop, here I am back again to the field, with fresh foods for stay up to date in the cutting edges of this Cas9 technology. Feast Easter reading this nice article :) 

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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-Cas System for Eukaryotic Genome Engineering | 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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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-Cas System for Eukaryotic Genome Engineering | 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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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-Cas System for Eukaryotic Genome Engineering | 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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RNA-guided gene activation by CRISPR-Cas9–based transcription factors

RNA-guided gene activation by CRISPR-Cas9–based transcription factors | CRISPR-Cas System for Eukaryotic Genome Engineering | 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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Addgene: Calarco Lab CRISPR Plasmids

Addgene: Calarco Lab CRISPR Plasmids | CRISPR-Cas System for Eukaryotic Genome Engineering | 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

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Genome editing in human pluripotent stem cells | StemBook

Genome editing in human pluripotent stem cells | StemBook | CRISPR-Cas System for Eukaryotic Genome Engineering | 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.

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Jun Liu's curator insight, July 4, 2013 11:50 PM

TALEN libray!

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Powerful gene-editing tool appears to cause off-target mutations in human cells

Powerful gene-editing tool appears to cause off-target mutations in human cells | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

A team of Massachusetts General Hospital researchers has found a significant limitation to the use of CRISPR-Cas RGNs, production of unwanted DNA mutations at sites other than the desired target, which indicates a need to improve the specificity of...

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CRISPRs extending their reach: prokaryotic RNAi protein Cas9 recruited for gene regulation : Article : The EMBO Journal

CRISPRs extending their reach: prokaryotic RNAi protein Cas9 recruited for gene regulation : Article : The EMBO Journal | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it
A Cas protein from the CRISPR defence system against foreign DNA, also functions in endogenous gene regulation. Sampson et al (2013) have revealed that in pathogenic Francisella bacteria, the Cas9 protein guided by small RNAs represses the mRNA of a lipoprotein. This novel mechanism of post-transcriptional regulation enables the infecting bacteria to evade the TLR2-based innate immune response of its host. Thus, reminiscent of eukaryotic RNAi where some proteins facilitate both genome defence and gene regulation, a central prokaryotic RNAi protein not only destroys invading DNA but also controls mRNA expression.
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Clem Stanyon's curator insight, June 17, 2013 4:18 AM

Many years ago, I attended a seminar given by one of the leaders in mouse genome manipulation, who had been experimenting with alternative ways to get foreign DNA inside an egg. As something of an aside, which often happens in science, his team discovered that inhibition of reverse transcriptase - that which makes DNA from RNA, which is the opposite direction to what normally happens and what retroviruses do in order to hijack infected cells - aborts development if that inhibition occurs before the 4-cell stage. This pretty much demonstrated that modifications of genomic DNA structure are essential to the initiation of new life in mammalian cells.

This is a parallel circumstance, indicating that things involved in genome defense can also be involved in gene regulation, and begs the question of whether there is any real difference: to the protein involved, Cas9, the ultimate cellular outcome is not the issue; the issue is the basic enzymatic activity.

Clem Stanyon's comment, June 17, 2013 4:21 AM
Here's an article about the wider field, apropos of the protein fold that is involved in the larger family of enzymes, which are all involved with variations on the same higher-structural field:
http://7thspace.com/headlines/439526/comprehensive_analysis_of_the_hepn_superfamily_identification_of_novel_roles_in_intra_genomic_conflicts_defense_pathogenesis_and_rna_processing.html
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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 | CRISPR-Cas System for Eukaryotic Genome Engineering | 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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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. | CRISPR-Cas System for Eukaryotic Genome Engineering | 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.


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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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Addgene: Qi Lab CRISPR Page

Addgene: Qi Lab CRISPR Page | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

Qi Lab CRISPR Plasmids Available from Addgene

 

A system to regulate a specific gene's expression based on the Type II bacterial CRISPR system for use in either human cells or bacterial cells that is called CRISPR Interference (CRISPRi). For use in human cells a human codon-optimized but enzymatically dead Cas9 nuclease can be targeted to a DNA sequence using a chimeric gRNA. The inactive Cas9/gRNA complex bound to DNA has been shown to interfere with transcriptional elongation, RNA polymerase binding and transcription factor binding. A BFP-Cas9 (enzymatically inactive) fusion construct was also created to help determine successful transformants. For use in bacteria a catalytically dead Cas9 expression plasmid and separate gRNA expression plasmid were also created. In addition, a wild type bacterial Cas9 allows for genome editing in bacterial cells.

 

These plasmids are described in:

Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression.Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.. PUBMED.

 

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A CRISPR/Cas system mediates bacterial innate immune evasion and virulence

A CRISPR/Cas system mediates bacterial innate immune evasion and virulence | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

Sampson et al., Nature

 

CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids1, 2, 3, 4. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation5, 6, 7, 8. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes9, 10. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens11, 12, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.

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Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

Jao et al., 2013, PNAS

 

A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75–99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.

 
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Clem Stanyon's curator insight, August 10, 2013 11:24 PM

With a bit of tinkering - modifying condon usage to better match that of the zebrea fish - the Cas9 protein would be expressed more effectively, which is probably how these researchers achieves such extremely high levels of targeted mutagenesis: 75-99%. This would also be the level needed to be able to modify the bulk of somatic tissue to overcome a dominant-negative allele, or to modify the genome of tetra- and hexa-ploid genomes.

BigField GEG Tech's curator insight, May 6, 2014 11:20 AM

http://geg-tech.com

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Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease

Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

Nekrasov et al., 2013, Nat Biotech

 

Sustainable intensification of crop production is essential to ensure food demand is matched by supply as the human population continues to increase1. This will require high-yielding crop varieties that can be grown sustainably with fewer inputs on less land. Both plant breeding and genetic modification (GM) methods make

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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-Cas System for Eukaryotic Genome Engineering | 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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CRISPR based genome editing - the future of molecular biology.

CRISPR based genome editing - the future of molecular biology. | CRISPR-Cas System for Eukaryotic Genome Engineering | 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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CRISPR RNA–guided activation of endogenous human genes

CRISPR RNA–guided activation of endogenous human genes | CRISPR-Cas System for Eukaryotic Genome Engineering | 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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Cell Reports - Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System

Cell Reports - Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

Bassett et al., 2013

Summary

Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated). We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.

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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 | CRISPR-Cas System for Eukaryotic Genome Engineering | 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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Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications


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Clem Stanyon's curator insight, June 17, 2013 4:31 AM

This isn't quite "genome engineering," but could readily fall into the category of "epigenome engineering", which is of particular interest to anyone looking into the basis of obesity, for example, or any other epi-genetic contidition. Such conditions are established by the environment, some duing gestation, some by early or recent exposure to dietary or other environmental factors.

Clem Stanyon's comment, June 17, 2013 5:32 AM
And here's a site that might contain more on the subject of epigenomic engineering:
http://epigenie.com/engineering-epigenomes-with-crispr-cas/
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Cell Press

Cell Press | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it
sign up for 27 June @CellStemCell webinar on genome editing in stem cells (TALENs, CRISPRs, etc) http://t.co/FKVHvKyZlv #yam

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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-Cas System for Eukaryotic Genome Engineering | 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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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 | CRISPR-Cas System for Eukaryotic Genome Engineering | Scoop.it

Wang et al., 2013, Cell

 

Highlights

---------------

> CRISPR/Cas9-mediated simultaneous targeting of five genes in mES cells

> Generation of Tet1/Tet2 double-mutant mice in one step

> Generation of Tet1/Tet2 double-mutant mice with predefined mutations in one step

 

Summary

--------------

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

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wb's curator insight, May 6, 2013 10:31 AM

amazing.....

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The New Genetic Engineering Toolbox - BioTechniques

(via T. Lahaye, thanks!)

comparison of TAL effectors, CRISPR/Cas9 and zinc fingers


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