Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign

J Am Chem Soc. 2014 Apr 2;136(13):5047-56. doi: 10.1021/ja4130059. Epub 2014 Mar 20.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • DNA / genetics
  • Directed Molecular Evolution / methods
  • Factor IX / genetics
  • Genome, Human
  • HEK293 Cells
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Engineering / methods*
  • Protein Multimerization
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombinases / chemistry*
  • Recombinases / genetics*
  • Recombinases / metabolism
  • Recombination, Genetic*
  • Zinc Fingers*
  • alpha-Galactosidase / genetics

Substances

  • Recombinant Fusion Proteins
  • Recombinases
  • Factor IX
  • DNA
  • alpha-Galactosidase