Towards a More Robust Salmon: Using CRISPR/Cas9 editing to increase protein levels in Zebrafish and Atlantic salmon

Abstract

With an increasing world population, the demand for high quality food grows every year. The aquaculture industry shows promising potential as it provides nutritionally rich, affordable provisions, but is still facing challenges such as high mortality rates in the fish populations. One possibility to confront this issue is to improve fish robustness through genome editing. A potential strategy could be to enhance the immune response by modulating gene regulatory mechanisms involved in the immune system function. MicroRNAs, a type of RNA element, represent one target candidate mechanism. MicroRNAs bind specific sites in the 3’ UTR of mRNA, ultimately initiating mRNA destabilizing actions. In this project, we performed genome editing by microinjection of both CRISPR/Cas9 and the cytosine base editor AncBE4max in newly fertilized zebrafish eggs. The aim was to edit miRNA binding sites in the mRNA 3’UTR of immune related transcripts, and thereby increase mRNA levels as the altered sites would be unrecognizable to the miRNA. The genes irf7, gata6 and nod2 were identified and chosen from a list of genes as the most suitable candidates based on a list of criteria. Validation of the mutagenesis was performed through the T7 endonuclease 1 assay, sanger sequencing and deep sequencing (Miseq). To measure relative changes in mRNA levels of the targeted genes, qPCR was performed utilizing injected five days old zebrafish larvae. CRISPR guides designed in this project targeting irf7 and nod2 were shown to have an extremely high efficiency approaching 100% editing frequencies. The sequencing analysis did not show changes in gata6 after injection of the AncBE4max. Miseq results showed a diverse assortment of deletions and insertions in the irf7 and nod2 mutated larvae, where deletions appeared to be the preferred method of reparations. Specific mutations were observed across separate individual larvae. Inserted templates identical to regions within close approximation to the cut sites were also observed as a response to fix the strand breaks. Significant decreases in relative mRNA levels were measured for both irf7 (56.27%) and nod2 (12.21%) mutated larvae. The findings of this research indicate the possibility of modulating mRNA levels through genome editing of the 3’ UTR.