Enhancing
the Specificity of Recombinase-Mediated
Genome Engineering through Dimer Interface Redesign
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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 Cys<sub>2</sub>-His<sub>2</sub> 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