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Coupling endonucleases with DNA end–processing enzymes to drive gene disruption

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Abstract

Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end–processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.

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Figure 1: Coupling endonucleases to exonucleases increases gene disruption.
Figure 2: DNA end–processing enzymes library screen.
Figure 3: Trex2 increases knockout of endogenous CCR5 with an engineered homing endonuclease.

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Acknowledgements

M.T.C. was supported in part by the US Public Health Service and National Research Service Award (T32 GM07270) from the US National Institute of General Medical Sciences. Additional funding was from the US National Institutes of Health (RL1CA133832, UL1DE019582, R01-HL075453, PL1-HL092557, RL1-HL092553, RL1-HL92554 and U19-AI96111) and Seattle Children's Center for Immunity and Immunotherapies. We would like to thank J. Stark (Beckman Research Institute and City of Hope) and D. Stetson (University of Washington) for initial Trex2 expression plasmids and D. Voytas (University of Minnesota) for TALEN cloning reagents. Zinc-finger nuclease expression plasmids were provided by C. Ramirez and K. Joung (Harvard University and Massachusetts General Hospital). J. Jarjour (Seattle Children's Research Institute, presently Pregenen Inc.) designed the Sce-SCID mouse, and Sce-SCID MEFs were provided by A. Astrakhan and G. Metzler (University of Washington). We thank all members of the Northwest Genome Editing Consortium for their many insightful discussions.

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Author notes

  1. Michael T Certo and Kamila S Gwiazda: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, USA

    Michael T Certo, Kamila S Gwiazda & Kyle Jacoby

  2. Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA

    Michael T Certo, Kamila S Gwiazda, Ryan Kuhar, Blythe Sather, Gabrielle Curinga, Tyler Mandt, Michelle Brault, Abigail R Lambert, Sarah K Baxter, Kyle Jacoby, Byoung Y Ryu, David J Rawlings & Andrew M Scharenberg

  3. Department of Immunology, University of Washington, Seattle, Washington, USA

    Sarah K Baxter, David J Rawlings & Andrew M Scharenberg

  4. Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Hans-Peter Kiem

  5. Department of Medicine, University of Washington, Seattle, Washington, USA

    Hans-Peter Kiem

  6. Department of Pathology, University of Washington, Seattle, Washington, USA

    Hans-Peter Kiem

  7. Cellectis, Paris, France

    Agnes Gouble, Frederic Paques & Andrew M Scharenberg

  8. Department of Pediatrics, University of Washington, Seattle, Washington, USA

    David J Rawlings & Andrew M Scharenberg

Authors
  1. Michael T Certo
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  2. Kamila S Gwiazda
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  3. Ryan Kuhar
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  4. Blythe Sather
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  5. Gabrielle Curinga
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  6. Tyler Mandt
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  7. Michelle Brault
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  8. Abigail R Lambert
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  9. Sarah K Baxter
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  10. Kyle Jacoby
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  11. Byoung Y Ryu
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  12. Hans-Peter Kiem
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  13. Agnes Gouble
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  14. Frederic Paques
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  15. David J Rawlings
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  16. Andrew M Scharenberg
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Contributions

M.T.C. designed and conceived experiments. M.T.C., K.S.G., R.K., B.S., G.C., M.B. and T.M. performed experiments. A.R.L., S.K.B., K.J., M.B., B.Y.R., H.-P.K., A.G. and F.P. provided reagents. M.T.C. wrote the paper and K.S.G., D.J.R. and A.M.S. edited the paper. D.J.R. and A.M.S provided technical support and conceptual advice.

Corresponding authors

Correspondence to David J Rawlings or Andrew M Scharenberg.

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Competing interests

A.M.S., F.P. and A.G. are employees of Cellectis and receive salary and equity compensation. A.M.S. is cofounder and board member of Pregenen Inc., a genome engineering company. A provisional patent (no. 61/447,672) for coupling endonucleases with exonucleases for gene disruption has been filed by Seattle Children's Research Institute.

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Supplementary Figures 1–10, Supplementary Tables 1 and 2 and Supplementary Note 1 (PDF 1352 kb)

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Certo, M., Gwiazda, K., Kuhar, R. et al. Coupling endonucleases with DNA end–processing enzymes to drive gene disruption. Nat Methods 9, 973–975 (2012). https://doi.org/10.1038/nmeth.2177

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