Herpes ICP8 protein stimulates homologous recombination in human cells

Abstract

ABSTRACTRecombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red SynExo homologous recombination proteins Beta inEscherichia colihas approached 100% efficiency. While highly efficient inE. coli, recombineering using the Red SynExo in other organisms declines in efficiency roughly correlating with phylogenetic distance fromE. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus Type 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstitutedin vitroand shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a “humanized” version of Beta protein from phage λ. ICP8 significantly enhanced gene targeting rates in HEK 293 T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.SIGNIFICANCEGenome modification by recombineering using SynExo viral recombination proteins has transformed functional genomic analysis in bacteria. Single-stranded DNA (ssDNA) recombineering approaches 100% efficiency inE. coliusing Beta protein from bacteriophage lambda, but recombineering has not been extended to eukaryotic genomes. Efficient recombineering requires SynExos that co-evolved with a viral host, however SynExos are common to viruses infecting a variety of organisms, including humans. The ICP8 protein of Human Herpes virus Type 1 is a SynExo protein similar to Beta. In this pioneering study, Herpes ICP8 stimulated gene targeting in a human genome by homologous recombination while the bacterial virus Beta protein inhibited recombination in human cells. This is the first demonstration of host-specific recombineering in human cells using a human viral SynExo protein.