Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss)

Authors: Cleveland, Beth; YAMAGUCHI, GINNOSUKE - Hokkaido University; Radler, Lisa; SHIMIZU, MUNETAKA - Hokkaido University

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/17/2018
Publication Date: 10/30/2018
Citation: Cleveland, B.M., Yamaguchi, G., Radler, L.M., Shimizu, M. 2018. Editing the duplicated insulin-like growth factor binding protein-2b gene in rainbow trout (Oncorhynchus mykiss). Scientific Reports. 8:16054. https://doi.org/10.1038/s41598-018-34326-6.
DOI: https://doi.org/10.1038/s41598-018-34326-6

Interpretive Summary: Insulin-like growth factor (IGF)-I is a positive regulator of growth in animals. The IGF-I hormone is carried in blood by IGF binding proteins, the most abundant of which is IGFBP-2b, although how it regulates IGF-I signaling is unknown. Advancements in gene editing technologies, particularly using the CRISPR/Cas9 system, has expanded the capacity for genetic modification in many animals, including fish. We used this technique to disrupt expression of the IGFBP-2b gene and reduced the abundance of the IGFBP-2b protein to less than 10% of normal levels. These findings indicate the potential for gene editing to unravel IGF-related mechanisms in salmonids. Successful application of the CRISPR/Cas9 system in rainbow trout indicates that this technique is valuable for characterizing protein-level functions to identify genes that are central regulators of economically important traits.

Technical Abstract: In salmonids, the majority of circulating insulin-like growth factor-I (IGFI) is bound to IGF binding proteins (IGFBP), with IGFBP-2b being the most abundant in circulation. In this study we used CRISPR/Cas9 methodology to disrupt expression of a functional IGFBP-2b protein by co-targeting IGFBP-2b1 and IGFBP-2b2 subtypes for gene editing. Twenty four rainbow trout were produced with mutations in the IGFBP-2b1 and IGFBP-2b2 genes. Gene variants were characterized in individual fish by analyzing the sequence of PCR products amplified from target regions using next-generation sequencing (100,000X coverage). Mutant fish exhibited between 8 - 100% and 2 - 83% mutant reads for IGFBP-2b1 and IGFBP-2b2, respectively, with a positive correlation in gene mutation rate between individual fish. Analysis of IGFBP-2b protein abundance indicated reductions in plasma IGFBP-2b abundance to between 0.04 - 0.96-fold of wild type levels. Reductions in plasma IGFBP-2b abundance in individual fish were positively correlated with percent gene mutagenesis (P=0.002, R2=0.47). Body weight and plasma IGF-I concentrations exhibited weak correlations with gene mutagenesis. These findings indicate that a combination of gene editing using CRISPR/Cas9 and ligand blotting is a feasible approach for characterizing protein-level functions of duplicated IGFBP genes in salmonids and is useful to unravel IGF-related endocrine mechanisms.