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Authors: Oldfield, Lauren M and Grzesik, Peter and Voorhies, Alexander A and Alperovich, Nina and MacMath, Derek and Najera, Claudia D and Chandra, Diya Sabrina and Prasad, Sanjana and Noskov, Vladimir N and Montague, Michael G and Friedman, Robert M and Desai, Prashant J and Vashee, Sanjay

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, KOS(YA), 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.

Journal: Proceedings of the National Academy of Sciences of the United States of America
DOI: 10.1073/pnas.1700534114
Year: 2017

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