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


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.

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Figure 1: Architectures of Cas9 and FokI-dCas9 fusion variants.
Figure 2: Genomic DNA modification by fCas9, Cas9 nickase and wild-type Cas9.
Figure 3: DNA modification specificity of fCas9, Cas9 nickase, and wild-type Cas9.
Figure 4: Genomic DNA modification specificity of fCas9 and Cas9 nickase at genomic sites with highly similar off-target sites.

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J.P.G., D.B.T. and D.R.L. were supported by Defense Advanced Research Projects Agency HR0011-11-2-0003 and N66001-12-C-4207, US National Institutes of Health NIGMS R01 GM095501 (D.R.L.) and the Howard Hughes Medical Institute (HHMI). D.R.L. was supported as a HHMI Investigator. We thank R. McDonald for technical assistance and V. Pattanayak for helpful comments.

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Authors and Affiliations



J.P.G. and D.B.T. performed the experiments, designed the research, analyzed the data and wrote the manuscript. D.R.L. designed the research, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to David R Liu.

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Competing interests

The co-authors have filed a provisional patent application related to this work. D.R.L. is a consultant for Editas Medicine, a company that applies genome editing technologies for human therapeutic applications.

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Supplementary Figures 1–11, Supplementary Results, Supplementary Notes and Supplementary Tables 1–5 (PDF 1127 kb)

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Guilinger, J., Thompson, D. & Liu, D. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol 32, 577–582 (2014).

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