Directed evolution of recombinase specificity by split gene reassembly

Charles A. Gersbach, Thomas Gaj, Russell M. Gordley, Carlos F. Barbas

Research output: Contribution to journalArticlepeer-review


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 enzymemediated reassembly of the gene encoding β-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 fingerrecombinase 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 randomlymutated 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.

Original languageEnglish (US)
Article numbergkq125
Pages (from-to)4198-4206
Number of pages9
JournalNucleic acids research
Issue number12
StatePublished - Feb 27 2010
Externally publishedYes

ASJC Scopus subject areas

  • Genetics


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