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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    • "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.
    Current issues in molecular biology 10/2015; 20:1-12. · 5.75 Impact Factor
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    • "Therefore, CRISPER/Cas9 in its original form is not compatible with ObLiGaRe. However, the newly developed dimeric CRISPR-FokI nuclease could be very useful as molecular scissors (Guilinger et al. 2014; Tsai et al. 2014), providing an alternative to ZFNs or TALENs for future applications of ObLiGaRe. ACKNOWLEDGMENTS We thank Edward Rebar, Philip Gregory, and the Sangamo BioSciences Production group for providing ZFNs and Fyodor Urnov for helpful comments. "
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    ABSTRACT: Targeted gene insertion is a goal of genome editing and has been done in cultured cells but only in a handful of whole organisms. The existing method to integrate foreign DNA using the homologous recombination pathway is inherently low efficiency, and many systems are refractory to this method. Several additional manipulations have been developed to gain higher efficiency by suppressing the competing dominant repair pathway of nonhomologous end-joining. However, this can be laborious and in practice limits the range of hosts where the method is applicable. Here, we use the preferred pathway of nonhomologous end-joining (used previously to create indels for gene inactivation) for precise integration of large DNA into the specified genomic target site of an intact animal. Our method uses site-specific cleavage, end-capture of cohesive ends and obligate ligation-gated recombination. This approach is straight-forward and yields high efficiency without additional gene manipulations; therefore it is easily applicable to a much broader range of organisms. We demonstrate its application to the fungus fly Sciara coprophila where a transformation system has not existed before. We integrated a 6.5 kb transgene precisely at the desired genomic target site of Sciara using this method. This provides the foundation for future experiments to explore the unique genetic features of this organism. Similarly, the method described here will allow insertion of large pieces of DNA into a diverse group of organisms for studies of their genetic attributes. Copyright © 2015 Author et al.
    G3-Genes Genomes Genetics 07/2015; 5(9). DOI:10.1534/g3.115.019901 · 3.20 Impact Factor
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    • "Additional studies will also be required to evaluate the specificity and toxicity of RNA-guided DNA endonucleases in vitro and in vivo. Recent developments, in which an inactivated Cas element was conjugated to the FokI endonuclease, that requires dimer formation is promising as thereby a higher specificity can be achieved (Tsai et al. 2014; Guilinger et al. 2014). Biophysical and biochemical studies on CRISPRs could help to improve the design of next-generation genome editing tools. "
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    ABSTRACT: Molecular scissors (MS), incl. Zinc Finger Nucleases (ZFN), Transcription-activator like endoncleases (TALENS) and meganucleases possess long recognition sites and are thus capable of cutting DNA in a very specific manner. These molecular scissors mediate targeted genetic alterations by enhancing the DNA mutation rate via induction of double-strand breaks at a predetermined genomic site. Compared to conventional homologous recombination based gene targeting, MS can increase the targeting rate 10,000-fold, and gene disruption via mutagenic DNA repair is stimulated at a similar frequency. The successful application of different MS has been shown in different organisms, including insects, amphibians, plants, nematodes, and mammals, including humans. Recently, another novel class of molecular scissors was described that uses RNAs to target a specific genomic site. The CRISPR/Cas9 system is capable of targeting even multiple genomic sites in one shot and thus could be superior to ZFNs or TALEN, especially by its easy design. MS can be successfully employed for improving the understanding of complex physiological systems, producing transgenic animals, incl. creating large animal models for human diseases, creating specific cell lines, and plants, and even for treating human genetic diseases. This review provides an update on molecular scissors, their underlying mechanism and focuses on new opportunities for generating genetically modified farm animals.
    Transgenic Research 01/2015; 24(3). DOI:10.1007/s11248-015-9862-z · 2.32 Impact Factor
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