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

Published on Jun 1, 2014in Nature Biotechnology31.86
· DOI :10.1038/nbt.2908
Shengdar Q. Tsai25
Estimated H-index: 25
Nicolas Wyvekens3
Estimated H-index: 3
+ 6 AuthorsJ. Keith Joung62
Estimated H-index: 62
(Harvard University)
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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 recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. The cleavage activity of an RFN depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation and therefore show improved specificities relative to wild-type Cas9 monomers. Importantly, direct comparisons show that 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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