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Alteration of Cre recombinase site specificity by substrate-linked protein evolution

Nature Biotechnology volume 19, pages 1047–1052 (2001)Cite this article

Abstract

Directed molecular evolution was applied to generate Cre recombinase variants that recognize a new DNA target sequence. Cre was adapted in a three-stage strategy to evolve recombinases to specifically recombine the new site. This complex multicycle task was made feasible by an improved directed-evolution procedure that relies on placing the recombination substrate next to the recombinase coding region. Consequently, those DNA molecules carrying the coding region for a successful recombinase are physically marked by the action of that recombinase on the linked substrate and are easily retrieved from a large background of unsuccessful candidates by PCR amplification. We term this procedure substrate-linked protein evolution (SLiPE). The method should facilitate the development of new recombinases and other DNA-modifying enzymes for applications in genetic engineering, functional genomics, and gene therapy.

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Figure 1: Directed-evolution strategy.
Figure 2: Activity of Cre recombinase on different lox sites.
Figure 3: Recombinases assayed in E. coli.
Figure 4: Recombinases assayed in mammalian cells.
Figure 5: Mapping of Fre mutations into the Cre structure.

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References

  1. Plückthun, A., Schaffitzel, C., Hanes, J. & Jermutus, L. In vitro selection and evolution of proteins. Adv. Protein Chem. 55, 367–403 (2000).

    Article  Google Scholar 

  2. Arnold, F.H. & Volkov, A.A. Directed evolution of biocatalysts. Curr. Opin. Chem. Biol. 3, 54–59 (1999).

    Article  CAS  Google Scholar 

  3. Wilson, D.S. & Szostak, J.W. In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68, 611–647 (1999).

    Article  CAS  Google Scholar 

  4. Beaudry, A.A. & Joyce, G.F. Directed evolution of an RNA enzyme. Science 257, 635–641 (1992).

    Article  CAS  Google Scholar 

  5. Roberts, R.W. Totally in vitro protein selection using mRNA–protein fusions and ribosome display. Curr. Opin. Chem. Biol. 3, 268–273 (1999).

    Article  CAS  Google Scholar 

  6. Jermutus, L., Ryabova, L.A. & Plückthun, A. Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 9, 534–548 (1998).

    Article  CAS  Google Scholar 

  7. Tawfik, D.S. & Griffiths, A.D. Man-made cell-like compartments for molecular evolution. Nat. Biotechnol. 16, 652–656 (1998).

    Article  CAS  Google Scholar 

  8. Buchholz, F., Angrand, P.O. & Stewart, A.F. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat. Biotechnol. 16, 657–662 (1998).

    Article  CAS  Google Scholar 

  9. Hoess, R., Abremski, K., Irwin, S., Kendall, M. & Mack, A. DNA specificity of the Cre recombinase resides in the 25 kDa carboxyl domain of the protein. J. Mol. Biol. 216, 873–882 (1990).

    Article  CAS  Google Scholar 

  10. Guo, F., Gopaul, D.N. & van Duyne, G.D. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389, 40–46 (1997).

    Article  CAS  Google Scholar 

  11. Hoess, R.H. & Abremski, K. Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J. Mol. Biol. 181, 351–362 (1985).

    Article  CAS  Google Scholar 

  12. Hoess, R.H., Wierzbicki, A. & Abremski, K. The role of the loxP spacer region in P1 site-specific recombination. Nucleic Acids Res. 14, 2287–2300 (1986).

    Article  CAS  Google Scholar 

  13. Rodriguez, C.I. et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25, 139–140 (2000).

    Article  CAS  Google Scholar 

  14. Farley, F.W., Soriano, P., Steffen, L.S. & Dymecki, S.M. Widespread recombinase expression using FLPeR (Flipper) mice. Genesis 28, 106–110 (2000).

    Article  CAS  Google Scholar 

  15. Drake, J.W. & Holland, J.J. Mutation rates among RNA viruses. Proc. Natl. Acad. Sci. USA 96, 13910–13913 (1999).

    Article  CAS  Google Scholar 

  16. Dorgai, L., Yagil, E. & Weisberg, R.A. Identifying determinants of recombination specificity: construction and characterization of mutant bacteriophage integrases. J. Mol. Biol. 252, 178–188 (1995).

    Article  CAS  Google Scholar 

  17. Yagil, E., Dorgai, L. & Weisberg, R.A. Identifying determinants of recombination specificity: construction and characterization of chimeric bacteriophage integrases. J. Mol. Biol. 252, 163–177 (1995).

    Article  CAS  Google Scholar 

  18. Hennighausen, L. & Furth, P.A. The right time and place for molecular scissors. Nat. Biotechnol. 17, 1062–1063 (1999).

    Article  CAS  Google Scholar 

  19. Metzger, D. & Feil, R. Engineering the mouse genome by site-specific recombination. Curr. Opin. Biotechnol. 10, 470–476 (1999).

    Article  CAS  Google Scholar 

  20. Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109 (2000).

    Article  CAS  Google Scholar 

  21. Seibler, J., Schübeler, D., Fiering, S., Groudine, M. & Bode, J. DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs. Biochemistry 37, 6229–6234 (1998).

    Article  CAS  Google Scholar 

  22. Soukharev, S., Miller, J.L. & Sauer, B. Segmental genomic replacement in embryonic stem cells by double lox targeting. Nucleic Acids Res. 27, e21 (1999).

    Article  CAS  Google Scholar 

  23. Lee, G. & Saito, I. Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216, 55–65 (1998).

    Article  CAS  Google Scholar 

  24. Feng, Y.Q. et al. Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. J. Mol. Biol. 292, 779–785 (1999).

    Article  CAS  Google Scholar 

  25. Lauth, M., Moerl, K., Barski, J.J. & Meyer, M. Characterization of Cre-mediated cassette exchange after plasmid microinjection in fertilized mouse oocytes. Genesis 27, 153–158 (2000).

    Article  CAS  Google Scholar 

  26. Thyagarajan, B., Guimarães, M.J., Groth, A.C. & Calos, M.P. Mammalian genomes contain active recombinase recognition sites. Gene 244, 47–54 (2000).

    Article  CAS  Google Scholar 

  27. Thyagarajan, B., Olivares, E.C., Hollis, R.P., Ginsburg, D.S. & Calos, M.P. Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Mol. Cell. Biol. 21, 3926–3934 (2001).

    Article  CAS  Google Scholar 

  28. Guo, H. et al. Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science 289, 452–457 (2000).

    Article  CAS  Google Scholar 

  29. Satoh, W., Hirai, Y., Tamayose, K. & Shimada, T. Site-specific integration of an adeno-associated virus vector plasmid mediated by regulated expression of rep based on Cre-loxP recombination. J. Virol. 74, 10631–10638 (2000).

    Article  CAS  Google Scholar 

  30. Logie, C. & Stewart, A.F. Ligand-regulated site-specific recombination. Proc. Natl. Acad. Sci. USA 92, 5940–5944 (1995).

    Article  CAS  Google Scholar 

  31. Goff, S.P. Genetics of retroviral integration. Annu. Rev. Genet. 26, 527–544 (1992).

    Article  CAS  Google Scholar 

  32. Russ, A.P., Friedel, C., Grez, M. & von Melchner, H. Self-deleting retrovirus vectors for gene therapy. J. Virol. 70, 4927–4932 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Choulika, A., Guyot, V. & Nicolas, J.F. Transfer of single gene-containing long terminal repeats into the genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP site. J. Virol. 70, 1792–1798 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Salmon, P. et al. Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes. Mol. Ther. 2, 404–414 (2000).

    Article  CAS  Google Scholar 

  35. Habu, Y. et al. Development of an HIV-1-dependent expression vector with the Cre/loxP system. Nucleic Acids Symp. Ser. 42, 295–296 (1999).

    Article  CAS  Google Scholar 

  36. Flowers, C.C., Woffendin, C., Petryniak, J., Yang, S. & Nabel, G.J. Inhibition of recombinant human immunodeficiency virus type 1 replication by a site-specific recombinase. J. Virol. 71, 2685–2692 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Lee, Y. & Park, J. A novel mutant loxP containing part of long terminal repeat of HIV-1 in spacer region: presentation of possible target site for antiviral strategy using site-specific recombinase. Biochem. Biophys. Res. Commun. 253, 588–593 (1998).

    Article  CAS  Google Scholar 

  38. Kim, S.T., Kim, G.W., Lee, Y.S. & Park, J.S. Characterization of Cre-loxP interaction in the major groove: hint for structural distortion of mutant Cre and possible strategy for HIV-1 therapy. J. Cell Biochem. 80, 321–327 (2001).

    Article  CAS  Google Scholar 

  39. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    Article  CAS  Google Scholar 

  40. Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).

    Article  CAS  Google Scholar 

  41. Zhao, H., Giver, L., Shao, Z., Affholter, J.A. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261 (1998).

    Article  CAS  Google Scholar 

  42. Buchholz, F., Ringrose, L., Angrand, P.O., Rossi, F. & Stewart, A.F. Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucleic Acids Res. 24, 4256–4262 (1996).

    Article  CAS  Google Scholar 

  43. Zhang, Y. et al. Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res. 24, 543–548 (1996).

    Article  CAS  Google Scholar 

  44. Gopaul, D.N., Guo, F. & Van Duyne, G.D. Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J. 17, 4175–4187 (1998).

    Article  CAS  Google Scholar 

  45. Guo, F., Gopaul, D.N. & Van Duyne, G.D. Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse. Proc. Natl. Acad. Sci. USA 96, 7143–7148 (1999).

    Article  CAS  Google Scholar 

  46. Kellendonk, C. et al. Regulation of Cre recombinase activity by the synthetic steroid RU 486. Nucleic Acids Res. 24, 1404–1411 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Mike Bishop for support, Mayte Pisabarro, Leonie Ringrose, Michael Huebner, and Yuri Voziyanov for discussions, and Michelle Meredyth for critical reading of the manuscript. This work was supported by funds from the National Institutes of Health (CA 44338), the G.W. Hooper Research Foundation, and the VW Foundation, Program on Conditional Mutagenesis.

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Authors and Affiliations

  1. Hooper Research Foundation, University of California San Francisco (UCSF), 513 Parnassus Ave., San Francisco, 94143-0552, CA, USA

    Frank Buchholz

  2. Genomics, Technische Universität Dresden, c/o MPI-CBG, Pfotenhauerstr. 108, Dresden, 01307, Germany

    A. Francis Stewart

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Correspondence to Frank Buchholz.

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Buchholz, F., Stewart, A. Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat Biotechnol 19, 1047–1052 (2001). https://doi.org/10.1038/nbt1101-1047

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