Characterization of Cuzd1 expression and development of in vivo CRISPR technology in Atlantic salmon (Salmo salar L.)

Abstract

The Atlantic salmon (Salmo salar L.) is a commercially important species that exhibits an anadromous life cycle involving migratory movements between freshwater and marine environments. Understanding the fundamental biology of the Atlantic salmon, particularly during key life history transitions such as smoltification, is therefore of great importance. In light of modern biotechnology and the completed reference genome for Atlantic Salmon, we can now examine the parr-smolt transition using a hypothesis-free characterization of gene expression analyses. We hypothesized that CUB and Zona Pellucida-like Domain 1 (Cuzd1) gene played a critical role in changing gill physiology during smoltification. To test this hypothesis, the study had two major aims: 1) to assess Cuzd1 expression in the whole fish through ontogeny and in various tissues during smoltification; 2) to develop in vivo CRISPR/Cas9 tools in Atlantic salmon, including the design of novel guides to enable functional characterization of Cuzd1. There was a significant difference in gene expression of Cuzd1 between the organ development stage and eyed stage, hatching, fin development, mid- point sampling, and approaching external feeding. This could provide valuable insight into the gene's developmental patterns. The gene expression during smoltification in various tissues during smoltification also varied with statistically significant differences in the muscle, gill, and brain. This finding implies that the role of Cuzd1 during smoltification is not limited to the gill. Thus, Cuzd1 exhibited tissue-specific expression patterns and could shed light on the mechanisms involved in the physiological changes during smoltification. A successful knockout of the positive control Slc45a2, a gene that affects pigmentation, was achieved. However, no evidence of Cuzd1 knockout could be observed, and further investigation is needed to enable a proper assessment. The result of this study demonstrated strong potential of the tested methodology for improving our understanding of Atlantic salmon biology and contributing to the development of novel methods for genetic characterization. Future work could be aimed at improving the methodology, including the use of promiscuous gRNAs and more comprehensive methods for genetic characterization. This work represents a significant step towards understanding the complex developmental transition of smoltification in Atlantic salmon. Overall, this study expands our characterization of Cuzd1 expression in Atlantic salmon and reports the first successful in vivo CRISPR knockouts at UiT, The Arctic University of Norway.