Like an automobile assembly line, these discrete parts can be modified as independent modules with a variety of genetic tools in vitro, in E. It is an application of existing tools to engineer large virus genomes and uses yeast genetics to separate the genome into multiple parts. The synthetic genomics assembly method described herein has many potential advantages over the BAC-based system ( 9). If multiple changes in the genome are desired, each must be made sequentially, greatly increasing the timeframe of making mutant viruses. Furthermore, each single modification with the BAC-based system takes weeks to complete. In addition, the allelic exchange methods used to make changes in the herpesvirus BAC expose the whole genome to very active recombinases, which could potentially generate unwanted changes. However, several issues, including genome instability, regarding herpesvirus-BAC clones have been reported ( 7, 8). This method enabled the introduction of reverse genetics-the ability to engineer desired changes in the genome and assess the phenotypic consequences-to many herpesviruses that previously could not be easily manipulated ( 4, 6). Many herpesvirus genomes have now been cloned as BAC plasmids ( 4) and, once in Escherichia coli, can be modified by recombineering methods ( 5). ( 3) to clone the mouse cytomegalovirus genome using this system. The use of bacterial artificial chromosome (BAC) cloning technology significantly advanced the capacity of herpesvirus researchers to manipulate virus genomes, following the work of Messerle et al. 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. 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. 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. The virus derived from this yeast-assembled genome, KOS YA, replicated with kinetics similar to wild-type virus. 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. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome.

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.

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.

Open gen bank in serial cloner