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

By S. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden, V. Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, Keith Joung. 2014

971 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

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Citations in the corpus

Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation (2016, background): “, 2016), or precisely editing the genome (Komor et al., 2016; Tsai et al., 2014).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “In 2012, Doudna and Charpentier showed the first use of CRISPR/Cas9 to introduce DSBs in target DNA (Jinek et al., 2012).”
The new frontier of genome engineering with CRISPR-Cas9 (2014, neutral): “at a defined distance apart (149, 150); and shorter sgRNAs truncated by”
Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array (2016, background): “, 2015) or coexpression of a specialized nuclease cutting a small sequence between sgRNAs (Nissim et al., 2014; Tsai et al., 2014).”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “A similar strategy has been achieved by using paired nuclease-deactivated Cas9 (dCas9)-FokI–sgRNA complexes (153, 154).”