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Research Project: Countermeasures to Control and Eradicate Foreign Animal Diseases of Swine

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Title: CRISPR/Cas gene editing of a large DNA virus: African Swine Fever Virus

item Borca, Manuel
item BERGGREN, KEITH - Oak Ridge Institute For Science And Education (ORISE)
item RAMIREZ, ELIZABETH - University Of Connecticut
item VUONO, ELIZABETH - Oak Ridge Institute For Science And Education (ORISE)
item Gladue, Douglas

Submitted to: Bio-protocol
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/24/2018
Publication Date: 8/20/2018
Citation: Borca, M.V., Berggren, K., Ramirez, E., Vuono, E., Gladue, D.P. 2018. CRISPR/Cas gene editing of a large DNA virus: African Swine Fever Virus. Bio-protocol. 8(16).

Interpretive Summary: Making changes to viruses to the virus genome is an important aspect to study how viruses interact with the animals they infect. The difference between a live vaccine and an infectious virus often has to do with changes in the virus genome. In order to understand how a particular virus causes disease, and to make a rational vaccine, a researcher has to make genetic changes to the virus. For African swine fever virus making these changes has been very hard, and time consuming. However with the discovery of CRISPR-Cas9 a system to make genetic changes, this process has become easier in many instances. This paper highlights in detail the protocol to use of CRISPR-Cas9 in the field of African swine fever virus, that can be applied to other large dsDNA viruses, allowing the ability to allow researchers to make genetic changes in the viral genome using this technology.

Technical Abstract: Gene editing of large DNA viruses, such as African swine fever virus (ASFV), have traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are then able to purify our recombinant virus from the parental by limiting dilution and plaque purification. We previously reported comparing the traditional homologous recombination methodology with CRISPR/Cas9, which resulted in over a 4 log increase in recombination.