Introduction
Large-scale aquaculture of Atlantic salmon (Salmo salar) is severely impacted by the sea louse Lepeophtheirus salmonis, with major consequences for fish welfare and industry sustainability . Pacific salmon species exhibit markedly lower lice burdens , suggesting intrinsic differences in immune responses during early infection. Understanding the molecular mechanisms that underpin this resistance is a critical step toward a more Pacific-like response in Atlantic salmon. In previous work, we observed multiple genes differentially expressed between species, including macrophage mannose receptor C type 1a (mrc1a) (ENSSSAG00000073565), found to be 2.73-fold upregulated in Coho salmon endothelial cells at 36 h post-infection. We posit therefore, that boosting expression of mrc1a may enhance immune responses and reduce lice loads in S. salar. To test this, we assessed the global transcriptional effects of mrc1a modulation using a high-throughput CRISPR-based promoter screen in Atlantic Salmon Kidney (ASK) cells. Following in vitro validation, a 888 bp hsp8 promoter fragment was introduced upstream of mrc1a in fertilized S. salar eggs to enable stable overexpression in vivo.
Materials and Methods pPB TRE dCas9-VPR or pPB TRE dCas9-KRAB constructs were stably transfected into ASK cells to generate CRISPR modulation lines. Cells were transfected with gRNAs targeting regions 50 bp–1 kb upstream of the mrc1a coding sequence, followed by RNA extraction and RNA sequencing. For knock ins, fertilized eggs were injected at the singlecell stage with Cas9–gRNA complexes, insertion templates and a HDR enhancer. Embryos were incubated at 8 °C until sampling, when, insertion efficiency was assessed by PCR, and mrc1a upregulation was quantified by qPCR.
Results and Discussion
Stable CRISPR modulation cell lines streamlines experimental workflows, enabling highthroughput screening of large guide libraries relevant to commercially important traits . This approaches is particularly valuable in salmonids, where paralogs may mask phenotypes. Here dCas9based screening identified which genes transcriptionally downstream of mrc1a were impacted as a result of both activation and repression. In embryos, no correlation was observed between homology arm length and knock in success. Which highlights the importance of screening multiple templates for a given target. Use of an HDR enhancer substantially improved large insert efficiency, achieving up to 60%. lthough mrc1a expression varied due to mosaicism, knock-in groups consistently showed higher expression than controls, with several individuals reaching levels comparable to previous pHSP8-based studies . Here, we demonstrate the utility of high-throughput dCas systems for screening regulatory effects of industrially relevant genes. This approach provided insights for producing transgenic Atlantic salmon with high-efficiency, functional insertions and can be rapidly applied in the future to gene targets relevant in all fields of aquaculture. />
Acknowledgment
This work was completed as part of the CrispResist project funded by FHF
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