Directed evolution of recombinase specificity by split gene reassembly

Directed evolution of recombinase specificity by split gene reassembly

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-medi...

Directed evolution of the site specificity of Cre recombinase.

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 r...

Control over enzymatic activity by DNA-directed split enzyme reassembly.

Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants

The serine recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys₂-His₂ zinc-finger proteins results in the creation of engineered zinc-finger recombinases (ZFRs) capable of achieving targeted genetic modifications. The flexibility afforded by zinc-...

Reprogramming the Specificity of Sortase Enzymes by Directed Evolution