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Tracking genome engineering outcome at individual DNA breakpoints

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.

References

  1. 1

    Carr, P.A. & Church, G.M. Genome engineering. Nat. Biotechnol. 27, 1151–1162 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Pâques, F. & Duchateau, P. Meganucleases and DNA double-strand break-induced recombination: perspectives for gene therapy. Curr. Gene Ther. 7, 49–66 (2007).

    Article  Google Scholar 

  3. 3

    Durai, S. et al. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res. 33, 5978–5990 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Porteus, M.H. & Carroll, D. Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967–973 (2005).

    CAS  Article  Google Scholar 

  5. 5

    Caldecott, K.W. Single-strand break repair and genetic disease. Nat. Rev. Genet. 9, 619–631 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Shrivastav, M., De Haro, L.P. & Nickoloff, J.A. Regulation of DNA double-strand break repair pathway choice. Cell Res. 18, 134–147 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Branzei, D. & Foiani, M. Regulation of DNA repair throughout the cell cycle. Nat. Rev. Mol. Cell Biol. 9, 297–308 (2008).

    CAS  Article  Google Scholar 

  8. 8

    Cann, K.L. & Hicks, G.G. Regulation of the cellular DNA double-strand break response. Biochem. Cell Biol. 85, 663–674 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).

    Article  Google Scholar 

  10. 10

    Metzger, M.J., McConnell-Smith, A., Stoddard, B.L. & Miller, A.D. Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template. Nucleic Acids Res. 39, 926–935 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Bennardo, N., Cheng, A., Huang, N. & Stark, J.M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008).

    Article  Google Scholar 

  12. 12

    Stark, J.M., Pierce, A.J., Oh, J., Pastink, A. & Jasin, M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol. Cell. Biol. 24, 9305–9316 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Nagaraju, G., Hartlerode, A., Kwok, A., Chandramouly, G. & Scully, R. XRCC2 and XRCC3 regulate the balance between short- and long-tract gene conversions between sister chromatids. Mol. Cell. Biol. 29, 4283–4294 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Brenneman, M.A., Wagener, B.M., Miller, C.A., Allen, C. & Nickoloff, J.A. XRCC3 controls the fidelity of homologous recombination: roles for XRCC3 in late stages of recombination. Mol. Cell 10, 387–395 (2002).

    CAS  Article  Google Scholar 

  15. 15

    Guirouilh-Barbat, J., Rass, E., Plo, I., Bertrand, P. & Lopez, B.S. Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends. Proc. Natl. Acad. Sci. USA 104, 20902–20907 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Pierce, A.J., Johnson, R.D., Thompson, L.H. & Jasin, M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13, 2633–2638 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Aubert, M. et al. Successful targeting and disruption of an integrated reporter lentivirus using the engineered homing endonuclease Y2 I-AniI. PLoS ONE 6, e16825 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Sarkis, C., Philippe, S., Mallet, J. & Serguera, C. Non-integrating lentiviral vectors. Curr. Gene Ther. 8, 430–437 (2008).

    CAS  Article  Google Scholar 

  19. 19

    Bennardo, N., Gunn, A., Cheng, A., Hasty, P. & Stark, J.M. Limiting the persistence of a chromosome break diminishes its mutagenic potential. PLoS Genet. 5, e1000683 (2009).

    Article  Google Scholar 

  20. 20

    Kustikova, O.S. et al. Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood 102, 3934–3937 (2003).

    CAS  Article  Google Scholar 

  21. 21

    McConnell Smith, A. et al. Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc. Natl. Acad. Sci. USA 106, 5099–5104 (2009).

    CAS  Article  Google Scholar 

  22. 22

    Lee, G.S., Neiditch, M.B., Salus, S.S. & Roth, D.B. RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination. Cell 117, 171–184 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Takeuchi, R., Certo, M., Caprara, M.G., Scharenberg, A.M. & Stoddard, B.L. Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation. Nucleic Acids Res. 37, 877–890 (2009).

    CAS  Article  Google Scholar 

  24. 24

    Matsuoka, S. et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316, 1160–1166 (2007).

    CAS  Article  Google Scholar 

  25. 25

    Ma, Y. & Lieber, M.R. Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs. J. Biol. Chem. 277, 10756–10759 (2002).

    CAS  Article  Google Scholar 

  26. 26

    Kumar, A., Fernandez-Capetillo, O. & Carrera, A.C. Nuclear phosphoinositide 3-kinase β controls double-strand break DNA repair. Proc. Natl. Acad. Sci. USA 107, 7491–7496 (2010).

    CAS  Article  Google Scholar 

  27. 27

    Guirouilh-Barbat, J. et al. Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells. Mol. Cell 14, 611–623 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Słabicki, M. et al. A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. PLoS Biol. 8, e1000408 (2010).

    Article  Google Scholar 

  29. 29

    Pierce, A.J., Hu, P., Han, M., Ellis, N. & Jasin, M. Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev. 15, 3237–3242 (2001).

    CAS  Article  Google Scholar 

  30. 30

    Shu, X. et al. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804–807 (2009).

    Article  Google Scholar 

Download references

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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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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