title: Efficient Genome Manipulation by Variants of Site-Specific Recombinases R and TD.
abstract: Genome engineering benefits from the availability of DNA modifying enzymes that have different target specificities and have optimized performance in different cell types. This variety of site-specific enzymes can be used to develop complex genome engineering applications at multiple loci. Although eight yeast site-specific tyrosine recombinases are known, only Flp is actively used in genome engineering. To expand the pool of the yeast site-specific tyrosine recombinases capable of mediating genome manipulations in mammalian cells, we engineered and analyzed variants of two tyrosine recombinases: R and TD. The activity of the evolved variants, unlike the activity of the native R and TD recombinases, is suitable for genome engineering in Escherichia coli and mammalian cells. Unexpectedly, we found that R recombinase benefits from the shortening of its C-terminus. We also found that the activity of wild-type R can be modulated by its non-consensus "head" sequence but this modulation became not apparent in the evolved R variants. The engineered recombinase variants were found to be active in all recombination reactions tested: excision, integration, and dual recombinase-mediated cassette exchange. The analysis of the latter reaction catalyzed by the R/TD recombinase pair shows that the condition supporting the most efficient replacement reaction favors efficient TD-mediated integration reaction while favoring efficient R-mediated integration and deletion reactions.
authors.affiliation: School of Biosciences, Louisiana Tech University, 1 Adams Boulevard, Ruston, LA 71272, USA.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Voziyanova
authors.affiliation: School of Biosciences, Louisiana Tech University, 1 Adams Boulevard, Ruston, LA 71272, USA.
authors.forename: Rachelle P
authors.initials: RP
authors.lastname: Anderson
authors.affiliation: School of Biosciences, Louisiana Tech University, 1 Adams Boulevard, Ruston, LA 71272, USA.
authors.forename: Riddhi
authors.initials: R
authors.lastname: Shah
authors.affiliation: School of Biosciences, Louisiana Tech University, 1 Adams Boulevard, Ruston, LA 71272, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.affiliation: School of Biosciences, Louisiana Tech University, 1 Adams Boulevard, Ruston, LA 71272, USA. Electronic address: voziyan@latech.edu.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Voziyanov
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D008322:Mammals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013012:Sorbitol / Q000031:analogs & derivatives; D014439:Tyramine / Q000031:analogs & derivatives
chemical_list: D011994:Recombinant Proteins; D045522:Recombinases; C076628:tyramine-deoxysorbitol; D013012:Sorbitol; D014439:Tyramine
keywords: RMCE; genome engineering; protein engineering; site-specific recombination; tyrosine recombinases