Alteration of Cre recombinase site specificity by substrate-linked protein evolution.
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.
Buchholz
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Hooper Research Foundation, University of California San Francisco (UCSF), 513 Parnassus Ave., San Francisco, CA 94143-0552, USA. fraenk@itsa.ucsf.edu
Stewart
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AF
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases