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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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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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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DOI: https://doi.org/10.1038/nbt1101-1047
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