CRISPRized Immortality– Novel approaches to immortalize fish cell lines - 3Rs

Norwegian aquaculture relies on vaccination as its main preventive strategy to combat infectious diseases. However, due in part to limited knowledge of fish immune responses many of the current vaccines have suboptimal efficacy and do not prevent disease outbreaks in the field. To develop superior fish vaccines we need to have a better understanding of how fish immunity works. To achieve this, tools designed specifically to study fish immunity are badly needed. One important tool consists of cell lines i.e. fish cells grown in the lab where they can be used as understudies to live fish in a broad range of immunological studies. Further, novel technological advancements originally developed to study human immunology such as the CRISPR gene-editing techniques need to be adapted for use in different fish species. In CRIPRized immortality, we have developed several new Atlantic salmon cell lines, each originating from different types of cells. These cell lines will form a platform that we can use to study a range of immunological functions in the lab. To date members of the project group have also establish cell specific and efficient methodologies that effectively deliver gene modifying proteins into these fish cell lines. This system can be used to effectively study the function of key immune genes at the protein level. The addition of these tools to the "salmon tool box" stand to have a high impact on many aspects of fish health by facilitating studies into how the salmon immune response controls infections without having to do experiments on live fish.

Using selective approaches multiple novel Atlantic salmon cell lines, including cell lines derived from pre-sorted and immune stimulated leucocytes have been established. These cell lines provide versatile in vitro model systems that can be used to measure the impact of pathogens, adjuvants and immune stimulants on specific cell subsets reducing the number of live fish used in research. Further, the salmon toolbox has been expanded with the development of optimized transfection protocols for efficient delivery of various Cas9 proteins into Atlantic salmon cell lines and primary leucocytes. In addition, a vector-based CRISPR/Cas9 system have been established for efficient integration of constructs into the genome of two Atlantic salmon cell line. The optimized CRISPR method increases possibilities for efficient homology directed genome editing in salmonid cells, in particular for future applications aimed at generating stable cell lines expressing epitope tagged fusion proteins

Today the most important prophylactic strategy in the Norwegian aquaculture is vaccination. However, current vaccines do not prevent disease outbreaks in the field. Development of next-generation fish vaccines remains challenging due to the limited knowledge of fish protective responses and host-pathogen interactions. Thus, studies focused on elucidating protective immunity in fish are critical. Today these studies are often carried out using large groups of experimentally infected fish. In many studies cell lines can replace live fish. However, few salmon cell lines are available limiting the types of studies that can be performed in vitro. Active immortalization of primary cells is a common technique used to generate mammalian cell lines. Here we propose to take advantage of the recent advances in targeted gene editing and use CRISPR mediated homology directed repair (HDR) to immortalize salmon primary cells. This will be achieved by disrupting tumor suppressor genes and/or introducing immortalizing factors. The aim is to generate a versatile in vitro system that can be used in place of live fish, to study various key aspects of salmon immune responses. As a novel approach to generating anti-salmon specific monoclonal antibodies we will also use CRISR mediated HDR to introduce epitope tags into key immune genes, generating fusion proteins that can be detected by prevalidated antibodies. This will generate an in vitro system that can be used to study, in detail, the immunological functions of specific genes at the protein level. Combined, these studies will significantly reduce the number of animals used in research and help to refine necessary in vivo studies. If successful, our research will be immensely valuable in design of future vaccines and provide a critical tool to measure the effects of infection, immune stimulation and adjuvants on specific cell populations.