Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification

By J. Guilinger, David B. Thompson, David R. Liu. 2014

819 citations recorded by the source corpus.

Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ∼15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing.

Subfields: Molecular Biology, Plant Science

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Citations in the corpus

Target specificity of the CRISPR-Cas9 system (2014, positive): “It has been reported that RNA-guided FokI nuclease is at least four fold more specific than paired Cas9 nickase [69], likely due to FokI nuclease only functioning when dimerized whereas Cas9 nickase can cleave as a monomer [70].”
Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system (2015, methodology): “For example, PTG could be used with Cas9 nickase to improve targeting fidelity (13, 33, 34), or with deactivated Cas9 transcriptionalactivator or -repressor to manipulate multiple gene expression (35, 36).”
Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System1 (2014, background): “…of double Cas9 nickases (Ran et al., 2013), truncated guide RNAs (Fu et al., 2014), and a fusion of the Flavobacterium okeanokoites (Fok1) nuclease and a catalytically inactive Cas9 (dCas9-Fok1; Guilinger et al., 2014; Tsai et al., 2014), have dramatically reduced off targeting up to 5,000-fold.”
CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites (2014, neutral): “Conflict of interest: none declared.”
Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement (2017, background): “…including selecting highly specific target sequences, optimizing nuclease expression, using truncated sgRNAs, dCas9-FokI fusions, and paired Cas9-nickases (Fujii et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013; Cho et al., 2014; Fu et al., 2014; Guilinger 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).”
The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells (2016, neutral): “10 20 Current strategies to reduce CRISPR-Cas9 off-target effects include the use of partially inactivated Cas9 nickases 21,22 and a catalytically dead Cas9 fused to the FokI endonuclease domain, 23–25 both of which require the design and delivery of two gRNAs.”