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

Publisher Website
Google Scholar
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.