Transcriptomic and Phenotypic Analysis of CRISPR/Cas9-Mediated gluk2 Knockout in Zebrafish

Abstract

As a subtype of kainite receptors (KARs), GluK2 plays a role in the perception of cold in the periphery sensory neuron. However, the molecular mechanism for gluk2 on the cold stress in fish has not been reported. In this article, real-time PCR assays showed that gluk2 was highly expressed in the brain and eyes of adult zebrafish. To study the functions of gluk2, gene knockout was carried out using the CRISPR/Cas9 system. According to RNA-seq analysis, we selected the differentially expressed genes (DEGs) that had significant differences in at least three tissues of the liver, gill, intestine, skin, brain, and eyes. Gene Ontology (GO) enrichment analysis revealed that cry1ba, cry2, per1b, per2, hsp70.1, hsp70.2, hsp70l, hsp90aa1.1, hsp90aa1.2, hspb1, trpv1, slc27a1b, park2, ucp3, and METRNL were significantly enriched in the ‘Response to temperature stimulus’ pathway. Through behavioral phenotyping assay, the gluk2−/− larval mutant displayed obvious deficiency in cold stress. Furthermore, TUNEL (TdT-mediated dUTP Nick-End Labeling) staining proved that the gill apoptosis of gluk2−/− mutant was increased approximately 60 times compared with the wild-type after gradient cooling to 8 °C for 15 h. Overall, our data suggested that gluk2 was necessary for cold tolerance in zebrafish.