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Nat Biotechnol. 2014 Jun;32(6):569-76. doi: 10.1038/nbt.2908. Epub 2014 Apr 25.

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

Author information

1
1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [4] Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
2
1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
3
1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
4
1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

Abstract

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.

Comment in

PMID:
24770325
PMCID:
PMC4090141
DOI:
10.1038/nbt.2908
[Indexed for MEDLINE]
Free PMC Article

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