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Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods

Authors :
Michael G. Montague
Peter Grzesik
Sanjana Prasad
Lauren M. Oldfield
Sanjay Vashee
Robert Friedman
Claudia D. Najera
Nina Alperovich
Diya Sabrina Chandra
Vladimir N. Noskov
Derek MacMath
Alexander A. Voorhies
Prashant Desai
Source :
Proceedings of the National Academy of Sciences. 114
Publication Year :
2017
Publisher :
Proceedings of the National Academy of Sciences, 2017.

Abstract

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Details

ISSN :
10916490 and 00278424
Volume :
114
Database :
OpenAIRE
Journal :
Proceedings of the National Academy of Sciences
Accession number :
edsair.doi...........141d80373718d991a26f53ba9124d9df
Full Text :
https://doi.org/10.1073/pnas.1700534114