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A TALE nuclease architecture for efficient genome editing

Abstract

Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator–like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

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Figure 1: Structure and DNA-binding specificity of TALE proteins.
Figure 2: Activation of the endogenous human NTF3 gene by engineered TALE transcription factors.
Figure 3: Modification of the endogenous NTF3 locus in human cells.
Figure 4: Modification of endogenous human CCR5.
Figure 5: TALEN-mediated gene editing by HDR.
Figure 6: Base preferences of the five RVDs used in this study, as determined by averaging SELEX-derived base preferences for NT-L (Fig. 2e) and NT-R (Supplementary Fig. 5), as well as two additional designed TALEs “VEGF-1” and “CCR5-1” (Supplementary Fig. 10).

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Acknowledgements

We thank S. Abrahamson for critically reviewing this manuscript, and also E. Wolffe and D. Guschin for helpful comments and suggestions. We also thank S. Orlando, Y. Santiago, S. Lussier, A. Vincent and S. Lam for technical support.

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Contributions

J.C.M. and S.T. designed studies, performed experiments and analyzed data. G.Q., K.A.B., J.W., D.F.X., X.M., D.E.P., K.L.H., and G.J.C. performed studies. S.T, G.Q., E.L., S.J.H., G.P.D., L.Z., and I.A. developed new procedures and assembled constructs. F.D.U., H.S.Z, M.C.H., L.Z., P.D.G. and E.J.R. supervised studies and designed experiments. J.C.M., P.D.G. and E.J.R. wrote the manuscript.

Corresponding author

Correspondence to Edward J Rebar.

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All authors are current or past full-time employees of Sangamo BioSciences, Inc.

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Miller, J., Tan, S., Qiao, G. et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29, 143–148 (2011). https://doi.org/10.1038/nbt.1755

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