title: Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase.
abstract: The development of new methods for gene addition to mammalian genomes is necessary to overcome the limitations of conventional genetic engineering strategies. Although a variety of DNA-modifying enzymes have been used to directly catalyze the integration of plasmid DNA into mammalian genomes, there is still an unmet need for enzymes that target a single specific chromosomal site. We recently engineered zinc-finger recombinase (ZFR) fusion proteins that integrate plasmid DNA into a synthetic target site in the human genome with exceptional specificity. In this study, we present a two-step method for utilizing these enzymes in any cell type at randomly-distributed target site locations. The piggyBac transposase was used to insert recombinase target sites throughout the genomes of human and mouse cell lines. The ZFR efficiently and specifically integrated a transfected plasmid into these genomic target sites and into multiple transposons within a single cell. Plasmid integration was dependent on recombinase activity and the presence of recombinase target sites. This work demonstrates the potential for broad applicability of the ZFR technology in genome engineering, synthetic biology and gene therapy.
authors.affiliation: The Skaggs Institute for Chemical Biology, Department of Molecular Biology, CA, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
authors.forename: Thomas
authors.initials: T
authors.lastname: Gaj
authors.forename: Russell M
authors.initials: RM
authors.lastname: Gordley
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Mercer
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
mesh_terms: D000818:Animals; D002460:Cell Line; D018390:Gene Targeting; D015894:Genome, Human; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D045522:Recombinases; D019895:Transposases