title: Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign.
abstract: Despite recent advances in genome engineering made possible by the emergence of site-specific endonucleases, there remains a need for tools capable of specifically delivering genetic payloads into the human genome. Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are a class of reagents capable of achieving this. The utility of these enzymes, however, has been constrained by their low overall targeting specificity, largely due to the formation of side-product homodimers capable of inducing off-target modifications. Here, we combine rational design and directed evolution to re-engineer the serine recombinase dimerization interface and generate a recombinase architecture that reduces formation of these undesirable homodimers by >500-fold. We show that these enhanced recombinases demonstrate substantially improved targeting specificity in mammalian cells and achieve rates of site-specific integration similar to those previously reported for site-specific nucleases. Additionally, we show that enhanced recombinases exhibit low toxicity and promote the delivery of the human coagulation factor IX and α-galactosidase genes into endogenous genomic loci with high specificity. These results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications.
authors.affiliation: The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gaj
authors.forename: Shannon J
authors.initials: SJ
authors.lastname: Sirk
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Tingle
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Mercer
authors.forename: Mark C
authors.initials: MC
authors.lastname: Wallen
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004247:DNA / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods; D005164:Factor IX / Q000235:genetics; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045522:Recombinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D016335:Zinc Fingers; D000519:alpha-Galactosidase / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D045522:Recombinases; D005164:Factor IX; D004247:DNA; D000519:alpha-Galactosidase