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Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection


Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224–induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.

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Figure 1: In vitro selection for ZFN-mediated cleavage.
Figure 2: DNA cleavage sequence specificity profiles for CCR5-224 and VF2468 ZFNs.
Figure 3: Evidence for a compensation model of ZFN target site recognition.
Figure 4: ZFNs can cleave a large fraction of target sites with three or fewer mutations in vitro.

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This research was supported by US National Institutes of Health (NIH) R01 GM065400 (D.R.L.), Defense Advanced Research Projects Agency HR0011-11-2-0003 (D.R.L.), the Howard Hughes Medical Institute (D.R.L.), NIH R01 GM088040 (J.K.J.), NIH DP1 OD006862 (J.K.J.), and the Jim and Ann Orr Massachusetts General Hospital Research Scholar award (J.K.J.). V.P. was supported by an NIH training grant to the Harvard University Training Program in Molecular, Cellular and Chemical Biology. C.L.R. was supported by a National Science Foundation Graduate Research Fellowship and a Ford Foundation Predoctoral Fellowship. The Massachusetts General Hospital Neuroscience core facility, supported by NIH P30 NS045776, provided qPCR capabilities. We thank J. Carlson, B. Dorr, C. Pattanayak, D. Reyon, J. Sander and D. Thompson for helpful discussions, M. Goodwin for technical assistance, and M. Maeder (Massachusetts General Hospital) for mammalian cell ZFN expression plasmids.

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V.P. and C.L.R. performed the experiments, designed the research, analyzed the data and wrote the manuscript. J.K.J. and D.R.L. designed the research, analyzed the data and wrote the manuscript.

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Correspondence to David R Liu.

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The authors declare no competing financial interests.

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Supplementary Figures 1–15, Supplementary Tables 1–7, Supplementary Notes 1–2, Supplementary Protocols 1–9, Supplementary Data (PDF 5131 kb)

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Pattanayak, V., Ramirez, C., Joung, J. et al. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 8, 765–770 (2011).

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