title: Target-specific variants of Flp recombinase mediate genome engineering reactions in mammalian cells.
abstract: Genome engineering relies on DNA-modifying enzymes that are able to locate a DNA sequence of interest and initiate a desired genome rearrangement. Currently, the field predominantly utilizes site-specific DNA nucleases that depend on the host DNA repair machinery to complete a genome modification task. We show here that genome engineering approaches that employ target-specific variants of the self-sufficient, versatile site-specific DNA recombinase Flp can be developed into promising alternatives. We demonstrate that the Flp variant evolved to recombine an FRT-like sequence, FL-IL10A, which is located upstream of the human interleukin-10 gene, and can target this sequence in the model setting of Chinese hamster ovary and human embryonic kidney 293 cells. This target-specific Flp variant is able to perform the integration reaction and, when paired with another recombinase, the dual recombinase-mediated cassette exchange reaction. The efficiency of the integration reaction in human cells can be enhanced by 'humanizing' the Flp variant gene and by adding the nuclear localization sequence to the recombinase.
authors.affiliation: School of Biosciences, Louisiana Tech University, Ruston, LA, USA.
authors.forename: Riddhi
authors.initials: R
authors.lastname: Shah
authors.affiliation: School of Biosciences, Louisiana Tech University, Ruston, LA, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.affiliation: School of Biosciences, Louisiana Tech University, Ruston, LA, USA.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Voziyanova
authors.affiliation: School of Biosciences, Louisiana Tech University, Ruston, LA, USA.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Voziyanov
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003062:Codon; D003412:Cricetulus; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; C508609:IL10 protein, human; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C413663:fluorescent protein 583; D016753:Interleukin-10; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases
keywords: Flp; RMCE; gene targeting; genome engineering; site-specific recombination