An unbiased genome-wide analysis of zinc-finger nuclease specificity


Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.

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Figure 1: Trapping of IDLVs into ZFN-induced DNA DSBs.
Figure 2: Gene targeting using ZFNs and a homologous donor vector.
Figure 3: CLIS at genomic loci in ZFN-treated cells.
Figure 4: Validation of (nr)LAM-PCR identified off-target CLIS loci.
Figure 5: Comparative analysis of zinc-finger sequence specificity in vivo.


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We thank U. Abel for fruitful discussions. Funding was provided by the Deutsche Forschungsgemeinschaft (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium für Bildung und Forschung (iGene), by the VIth + VIIth Framework Programs of the European Commission (EC, European Network for the Advancement of Clinical Gene Transfer and Therapy (CLINIGENE) and Persisting Transgenesis (PERSIST) and by the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer to C.v.K. and M.S. Funding to L.N. was provided by Telethon (TIGET grant), EC (FP7-HEALTH-2009-222878, PERSIST; ERC Advanced Grant FP7, Targeting gene therapy - 249845).

Author information

R.G., A.L., H.G., M.S., J.C.M., P.D.G., M.C.H., L.N. and C.v.K. conceived the project, designed experiments and interpreted data. R.G., A.L., P.G., C.K., A.N., J.W., G.F. and C.C.B. performed experiments. R.G., A.A. and J.C.M. conducted bioinformatics analysis. M.C.H. and P.D.G. provided ZFN. R.G., A.L., A.A., J.C.M., M.C.H., P.D.G., M.S., L.N. and C.v.K. prepared and wrote the manuscript.

Correspondence to Luigi Naldini or Christof von Kalle.

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Competing interests

J.C.M., J.W., G.F., M.C.H. and P.D.G. are full-time employees of Sangamo BioSciences.

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Supplementary Tables 1,2, Supplementary Discussion, Supplementary Methods and Supplementary Figures 1–16 (PDF 927 kb)

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Gabriel, R., Lombardo, A., Arens, A. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol 29, 816–823 (2011).

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