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Proc Natl Acad Sci U S A. 2017 Oct 17;114(42):E8885-E8894. doi: 10.1073/pnas.1700534114. Epub 2017 Sep 19.

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

Author information

1
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, Rockville, MD 20850.
2
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University, Baltimore, MD 21231.
3
Department of Infectious Diseases, J. Craig Venter Institute, Rockville, MD 20850.
4
Policy Center, J. Craig Venter Institute, La Jolla, CA 92037.
5
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University, Baltimore, MD 21231; pdesai@jhmi.edu svashee@jcvi.org.
6
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, Rockville, MD 20850; pdesai@jhmi.edu svashee@jcvi.org.

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.

KEYWORDS:

genome-wide engineering; herpes simplex virus type 1; herpesvirus; synthetic genomics; tegument genes

Comment in

PMID:
28928148
PMCID:
PMC5651731
DOI:
10.1073/pnas.1700534114
[Indexed for MEDLINE]
Free PMC Article

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