Location: Foreign Animal Disease Research
Project Number: 3022-32000-063-018-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2020
End Date: May 31, 2022
Objective:
African swine fever is a large DNA virus of approximately 180kb and contains over 150 proteins; how the virus and virus proteins evade the host cellular immune response is unknown. Investigating which host proteins are involved in the immune response to the virus or the host proteins in which the virus utilizes to evade the immune response is critical to understand how the virus interacts with the host, and how different host-viral interactions occur in different cells of the host. Crisper knockout (CRISPR KO) libraries or Crisper activation (CRISPRa) libraries are a powerful way to do this, by targeting every protein in the genome for either reduced or increased expression. Doing this in several different stable cell types that are representative of the cell types that a virus would encounter during virus infection allows the ability to see what occurs with different populations of cells each expressing different CRISPR constructs targeted to different cellular proteins. ARS, PIADC and Vector builder (a licensed user of Crisper) will develop and test a series of libraries to develop a stable cell line which can support viral growth for use in vaccine development.
Approach:
To design and create the CRISPR KO and CRISPRa libraries first we will utilize a genomics approach of selecting specific sequences, called gRNAs for each gene in the genome to check for specificity and cross-reactivity. Once this is accomplished, we will then, by de novo synthesis, make all of the targeted sequences. These targeted sequences will be evaluated and used in a library approach; each unique and can be identified by next generation sequencing. Once this is accomplished, we will then express and grow these libraries and confirm for complexity of the libraries across the genome. We will then create a stable cell line that has high levels of expression for Cas9 the needed component for the CRISPR Library to function. Once we have adequate Cas9 expression, we will test for the ability of virus to grow in the host cell to confirm that Cas9 was inserted into a site that doesn’t affect virus growth. Once this is achieved we will then use both the CRISPR KO and CRISPRa libraries to transduce the Cas9 expressing cell lines to stably express these CRISPR libraries which in turn creates an in vivo library of cells each expressing theif own CRISPR targeted sequence that can be monitored by next-generation sequencing in order to determine which cells have a decreased or increased effect on viral replication. This process needs to be done with a diverse set of cell lines to determine how the virus interacts with different types of cells in the host. Once this process is achieved validation of the effectiveness of using CRISPR KO or CRISPERa in these cells will be determined by testing for the ability of virus to replicate in the individual cells. Then, by Next generation sequencing, we will determine which specific gene is being targeted in the cells to determine which host proteins are necessary for combating the virus, and which host proteins are necessary for the virus to evade the immune response.
