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

I norsk oppdrettsnæringen er vaksinasjon er det mest brukte sykdomsforebyggende tiltak, men på grunn av en manglende forståelse av fiskens immunforsvar går utviklingen av nye vaksiner langsomt. For å underlette flere studier av laksens immunforsvar trenger vi først og fremst flere og bedre verktøy, hvorav en av de største utfordringene er mangelen på dyrkbare laksecellelinjer. Videre må nye teknologiske fremskritt som opprinnelig ble utviklet for å studere immunsystemet hos mennesker, som CRISPR-genredigeringsteknikker, tilpasses slik at det kan brukes i ulike fiskearter. I prosjektet CRIPRized immortality har vi utviklet flere nye laksecellelinjer, som hver stammer fra ulike typer celler. Disse cellelinjene danner en plattform som vi kan bruke til å studere en rekke immunologiske funksjoner i laboratoriet. Medlemmar i projektgruppen har också etablerat cellspecifika och "laxe anpassade" metoder som effektivt levererar proteiner med gen modificerande functioner till dessa fiskcellinjer. Detta system kan användas för att effektivt studera funktionen hos viktiga immungener på proteinnivå. Tillägget av dessa verktyg till "laxverktygslådan" kommer att ha stor inverkan på många aspekter av fiskens hälsa genom att underlätta studier av hur laxens immunsvar kontrollerar infektioner utan att behöva göra experiment på levande fisk.

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.