Directed evolution of the site specificity of Cre recombinase

Proceedings of the National Academy of Sciences of the United States of America
Stephen W Santoro, Peter G Schultz

Abstract

Cre recombinase from bacteriophage P1 recognizes a 34-bp recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA. To better understand the molecular basis of protein-DNA recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxP sites. To be selected, members of a library of Cre variants produced by targeted random mutagenesis must rapidly catalyze recombination, in vivo, between two variant loxP sites that are located on a reporter plasmid. Recombination results in an altered pattern of fluorescent protein expression that can be identified by flow cytometry. Fluorescence-activated cell sorting can be used either to screen positively for recombinase variants that recognize a novel loxP site, or negatively for variants that cannot recognize the wild-type loxP site. The use of positive screening alone resulted in a relaxation of recombination site specificity, whereas a combination of positive and negative screening resulted in a switching of specificity. One of the identified recombinases selectively recombines a novel recom...Continue Reading

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