UMMS Affiliation

Department of Neurology

Publication Date

2018-08-15

Document Type

Article

Disciplines

Amino Acids, Peptides, and Proteins | Bacteria | Genetic Phenomena | Genetics and Genomics | Viruses

Abstract

Recombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red homologous recombination protein Beta in Escherichia coli has approached 100% efficiency. While highly efficient in E. coli, recombineering using the Red Synaptase/Exonuclease pair (SynExo) in other organisms declines in efficiency roughly correlating with phylogenetic distance from E. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstituted in vitro and 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 lambda. ICP8 significantly enhanced gene targeting rates in HEK 293T 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.

Keywords

Gene targeting, Recombinant proteins, DNA recombination, Homologous recombination, 293T cells, DNA repair, DNA replication, DNA annealing

Rights and Permissions

Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

DOI of Published Version

10.1371/journal.pone.0200955

Source

PLoS One. 2018 Aug 15;13(8):e0200955. doi: 10.1371/journal.pone.0200955. eCollection 2018. Link to article on publisher's site

Journal/Book/Conference Title

PloS one

Related Resources

Link to Article in PubMed

PubMed ID

30110337

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons 1.0 Public Domain Dedication.

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