Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol

Nature Biotechnology (Impact Factor: 41.51). 04/2014; 32(6). DOI: 10.1038/nbt.2908
Source: PubMed


Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

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Available from: Vishal Thapar, Sep 24, 2015
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    • "In the FokI-based CRISPR/Cas system, the FokI nuclease domain is fused to a catalytically-inactive Cas9 protein. After being recruited by two gRNAs, the dimers of the FokI fusion protein mediate sequence-specific DNA cleavage, with a defined spacing and orientation[55,60]. Quantitatively, the specificity of the FokI-based CRISPR/Cas was at least 140 fold higher than that of the wild type Cas9, and even fourfold higher than that of Cas9 nickase at similar endogenous off-target loci[60]. "
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    ABSTRACT: Technological advances are important for innovative biological research. Development of molecular tools for DNA manipulation, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas), has revolutionized genome editing. These approaches can be used to develop potential therapeutic strategies to effectively treat heritable diseases. In the last few years, substantial progress has been made in CRISPR/Cas technology, including technical improvements and wide application in many model systems. This review describes recent advancements in genome editing with a particular focus on CRISPR/Cas, covering the underlying principles, technological optimization, and its application in zebrafish and other model organisms, disease modeling, and gene therapy used for personalized medicine.
    Full-text · Article · Jan 2016
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    • "RFN cleavage activity strictly depends on the binding of two gRNAs onto target DNA. The DNA cleavage requires the PAM sites to be a certain distance apart and in a particular orientation with respect to each other (Tsai et al., 2014). "
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    ABSTRACT: CRISPR/Cas, a microbial adaptive immune system, has recently been reshaped as a versatile genome editing approach, endowing genome engineering with high efficiency and robustness. The DNA endonuclease Cas, a component of CRISPR system, is directed to specific target within genomes by guide RNA (gRNA) and performs gene editing function. However, the system is still in its infancy and facing enormous challenges such as off-target mutation. Lots of attempts have been made to overcome such off-targeting and proven to be effective. In this review we focused on recent progress of increasing the CRISPR specificity realized by rational design of gRNA and modification of Cas9 endonuclease. Meanwhile the methods to screen off-target mutation and their effects are also discussed. Comprehensive consideration and rational design to reduce off-target mutation and selection of effective screening assay will greatly facilitate to achieve successful CRISPR/Cas system mediated gene editing.
    Full-text · Article · Oct 2015 · Current issues in molecular biology
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    • "Modifications to the FokI domain have also been created to increase nuclease activity and specificity (Miller et al. 2007; Szczepek et al. 2007; Guo et al. 2010; Doyon et al. 2011b). Moreover, fusion of the FokI domain to the nuclease-inactivated Cas9 protein (dCas9) has also been used to increase the specificity of the CRISPR gene editing system (Guilinger et al. 2014b; Tsai et al. 2014). Optimizing the length of the ZF or TALE array can also increase nuclease activity and specificity (Bhakta et al. 2013; Guilinger et al. 2014a). "
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    ABSTRACT: Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances.
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