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

Abstract

<p id="d12969051e329">Viruses with large DNA genomes, such as herpesviruses, are difficult to manipulate with existing genetic tools. We describe an application of synthetic genomics assembly tools that enables rapid and efficient generation of combinatorial mutations in herpesvirus genomes. The method provides the capacity to design, generate, and test numerous multiloci mutants in parallel, which can help us understand basic virus biology, facilitate vaccine development, and aid development of next-generation virus-based delivery systems. This class of viruses is being used as vectors for therapeutics and vaccines, with an oncolytic herpesvirus approved for the treatment of melanoma. Although such improvements in genome assembly and manipulation raise dual-use concerns, we believe the potential benefits substantially outweigh the risks. </p><p class="first" id="d12969051e332">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 <i>Escherichia coli</i> 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. </p>