Knockout of EPO gene in blunt snout bream (Megalobrama amblycephala) by CRISPR/Cas9 reveals its roles in hypoxia-tolerance

Erythropoietin (EPO) is a glycoprotein hormone that plays a crucial role as a downstream regulator in the Hypoxia-inducible Factor (HIF) signaling pathway, mainly involved in the regulation of erythropoiesis. The open reading frame (ORF) of EPO in blunt snout bream (Megalobrama amblycephala, BSB) was 561 bp, encoding 186 amino acids. Phylogenetic analysis revealed it clusters with homologs in others teleost. We designed two target sites (targets 1, 2) to obtain gene knockout mutants, with the mature mRNA sequence of EPO that were located in exon 2, exon 3, respectively. The mutation efficiency of targets 1, 2 was 19.60%, 24.22%, respectively, both containing 6 mutation types. F1 EPO+/− mutants with the same mutation type were screened, and select F2 EPO−/− mutants after mating. At the age of five months, EPO−/− fish demonstrated a significant (p < 0.05) increase in the average oxygen tension threshold for the loss of equilibrium (LOEcrit) and the shedding degree of interlamellar cell masses (ILCM), whereas a significant (p < 0.05) decrease was observed in the number of red blood cells (RBC) and the concentration of hemoglobin (Hb) under hypoxia conditions, in comparison to the control group. Notably, injection of EPO mRNA rescued the downregulation of EPO expression in EPO−/− mutant, and overexpression of the EPO gene did not disturb the normal development of BSB embryos. These findings provide new insights into the potential for applying CRISPR/Cas9 in other aquaculture fish species and the breeding of hypoxia-tolerant BSB.