Abstract:
: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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Gersbach CA,Gaj T,Gordley RM,Barbas CF 3rddoi
10.1093/nar/gkq125subject
Has Abstractpub_date
2010-07-01 00:00:00pages
4198-206issue
12eissn
0305-1048issn
1362-4962pii
gkq125journal_volume
38pub_type
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