Directed evolution of recombinase specificity by split gene reassembly.

Published

Journal Article

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-mediated reassembly of the gene encoding beta-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.

Full Text

Duke Authors

Cited Authors

  • Gersbach, CA; Gaj, T; Gordley, RM; Barbas, CF

Published Date

  • July 2010

Published In

Volume / Issue

  • 38 / 12

Start / End Page

  • 4198 - 4206

PubMed ID

  • 20194120

Pubmed Central ID

  • 20194120

Electronic International Standard Serial Number (EISSN)

  • 1362-4962

International Standard Serial Number (ISSN)

  • 0305-1048

Digital Object Identifier (DOI)

  • 10.1093/nar/gkq125

Language

  • eng