University of Massachusetts Medical School Faculty Publications

UMMS Affiliation

Department of Neurology

Publication Date

2-11-2018

Document Type

Article Preprint

Disciplines

Genetic Phenomena | Genetics and Genomics | Molecular Biology | Virology

Abstract

Recombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red SynExo homologous recombination proteins Beta in Escherichia coli has approached 100% efficiency. While highly efficient in E. coli, recombineering using the Red 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 Type 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 λ. 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

Herpes ICP8 protein, Escherichia coli, Human Herpes virus Type 1, SynExo, ICP8 synaptase, UL12 exonuclease, recombineering, gene targeting, molecular biology

Rights and Permissions

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.

DOI of Published Version

10.1101/259739

Source

bioRxiv 259739; doi: https://doi.org/10.1101/259739. Link to preprint on bioRxiv service.

Journal/Book/Conference Title

bioRxiv

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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