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Tracking genome engineering outcome at individual DNA breakpoints

Nature Methods volume 8, pages 671–676 (2011)Cite this article

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Abstract

Site-specific genome engineering technologies are increasingly important tools in the postgenomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated 'traffic light', that supports rapid flow-cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high-throughput screens for factors that bias the engineering outcome. We applied the traffic light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred.

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Figure 1: The traffic light reporter.
Figure 2: Titration of nuclease and donor template.
Figure 3: Four-color system to track nuclease and donor template delivery simultaneously with the TLR.
Figure 4: Effect of single versus double-strand DNA breaks on engineering outcome.
Figure 5: High-throughput siRNA kinome screen to identify modifiers of engineering outcome.

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Acknowledgements

M.T.C. was supported in part by Public Health Service, National Research Service Award, T32 GM07270, from the US National Institute of General Medical Sciences. Additional funding was from US National Institutes of Health (RL1CA133832, UL1DE019582, R01-HL075453, PL1-HL092557 and RL1-HL092553) and Seattle Children's Center for Immunity and Immunotherapies. We thank C. Ramirez and K. Joung for zinc-finger nuclease constructs (Harvard University, Massachusetts General Hospital), and all members of the Northwest Genome Engineering Consortium for their many insightful discussions.

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Author notes

  1. Jordan Jarjour

    Present address: Present address: Precision Genome Engineering Inc., Seattle, Washington, USA.,

Authors and Affiliations

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

    Michael T Certo

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

    Michael T Certo, Byoung Y Ryu, Mikhail Garibov, Jordan Jarjour, David J Rawlings & Andrew M Scharenberg

  3. Quellos High Throughput Core, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA

    James E Annis

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

    Jordan Jarjour, David J Rawlings & Andrew M Scharenberg

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

    David J Rawlings & Andrew M Scharenberg

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Contributions

M.T.C. designed and performed experiments, analyzed data and wrote the paper; B.Y.R., J.E.A. and M.G. performed experiments; J.J. and D.J.R. designed experiments; and A.M.S. designed experiments and wrote the paper.

Corresponding authors

Correspondence to David J Rawlings or Andrew M Scharenberg.

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The authors declare no competing financial interests.

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Supplementary Figures 1–8, Supplementary Tables 1–2 and Supplementary Notes 1–2 (PDF 1345 kb)

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Certo, M., Ryu, B., Annis, J. et al. Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods 8, 671–676 (2011). https://doi.org/10.1038/nmeth.1648

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