Genetic Engineering of CRISPR
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CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering - Nature Biotech.

(via T. Lahaye, thx)

Mali et al (2013)

 

As a genome editing tool TAL effector–FokI dimers are usually used, and for genome regulation TAL effector–VP64 fusions have been shown to be highly effective. We used the latter as ... this format also reveals the specificity profile of individual TAL-effectors. Examining the TAL effector off-targeting data ... reveals that 18-mer TAL effectors can potentially tolerate 1 or 2 mutations in their target sequences, but fail to activate a large majority of three-base mismatch variants in their targets. They are also particularly sensitive to mismatches nearer
the 5ʹ end of their target sequences. Notably, certain mutations in the middle of the target lead to greater TAL effector activity, an aspect that needs further evaluation. We confirmed a subset of the above results through targeted experiments in a nuclease assay ... We also observed that shorter TAL effectors (14-mer and 10-mer) are progressively less tolerant of mismatches but also reduced in activity by an order of magnitude...
To decouple the role of individual repeat-variable di-residues (RVDs), we confirmed that choice of RVDs does contribute to base specificity but TAL effector specificity is also a function of the binding energy of the protein as a whole ... our data imply that engineering shorter TAL effectors or TAL effectors bearing a judicious composition of high- and low-affinity monomers can potentially yield higher specificity in genome engineering applications, and the requirement for FokI dimerization in nuclease applications enables a further dramatic reduction in off-target effects especially when using the shorter TAL effectors.


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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. | Genetic Engineering of CRISPR | 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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Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease

Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease | Genetic Engineering of CRISPR | 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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PLOS ONE: Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs)

PLOS ONE: Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs) | Genetic Engineering of CRISPR | Scoop.it

(suggested by Tom Schreiber, thx)

Hu et al, 2013

MicroRNAs (miRNAs) have quickly emerged as important regulators of mammalian physiology owing to their precise control over the expression of critical protein coding genes.

Despite significant progress in our understanding of how miRNAs function in mice, there remains a fundamental need to be able to target and edit miRNA genes in the human genome. Here, we report a novel approach to disrupting human miRNA genes ex vivo using engineered TAL-effector (TALE) proteins to function as nucleases (TALENs) that specifically target and disrupt human miRNA genes. We demonstrate that functional TALEN pairs can be designed to enable disruption of miRNA seed regions, or removal of entire hairpin sequences, and use this approach to successfully target several physiologically relevant human miRNAs including miR-155*, miR-155, miR-146a and miR-125b.

This technology will allow for a substantially improved capacity to study the regulation and function of miRNAs in human cells, and could be developed into a strategic means by which miRNAs can be targeted therapeutically during human disease.


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