CRISPR/Cas9 mutagenesis reveals an essential role of PI4KB in promoting growth and resisting hemorrhagic disease caused by GCRV-II infection in juvenile grass carp
Few studies reported for obtaining the grass carp resistant to hemorrhagic disease via gene editing in commercial fish. Here, we demonstrate that the expression and activity of grass carp PI4KB (gcPI4KB) are vital for GCRV-I and GCRV-II replication. Due that obvious cytopathic effect (CPE) in the present available cell lines is only caused by GCRV-I, but GCRV-II is the current popular and fatal strain in grass carp, GCRV-I and GCRV-II are used in cell lines and in grass carp, respectively. The in vitro studies in CIK cells revealed that gcPI4KB interacted with NS80 and VP3 of GCRV-I, and that gcPI4KB was recruited by NS80 for promoting the generation of GCRV VIBs. Since the negative regulatory role of gcPI4KB in GCRV infection was confirmed by in vitro data,we performed gene editing of gcPI4KB in grass carp. We found that PI4KB F0 crispants juvenile grass carp have obvious advantages in promoting growth and in resisting GCRV-II infection. Compared with uninfected WT grass carp, the uninfected PI4KB F0 crispants juvenile grass carp exhibit a higher expression level of many genes involved in growth- and development-related metabolic pathways such as the FoxO signaling pathway and insulin signaling pathway. Compared with WT grass carp without infection, PI4KB F0 crispants juvenile grass carp without infection or WT grass carp infected with GCRV-II, higher expression levels for many genes involved in metabolic diseases and viral infection were observed in the liver from PI4KB F0 crispants juvenile grass carp infected with GCRV-II. Altogether, the present study suggests the mechanism of gcPI4KB in facilitating GCRV replication, the signaling pathways regulated by gcPI4KB, and the possibility to obtain the grass carp resistant to hemorrhagic disease via gene editing of PI4KB. Overall design: The gcPI4KB F0 crispants were generated using CRISPR/Cas9 for gene-editing gcPI4KB in grass carp. In brief, the single guide RNA(sgRNA) of gcPI4KB was designed using the ZiFiT (http://zifit.partners.org/ZiFiT/). To explore the influence of gcPI4KB mutant on GCRV-II infection in vivo, the 6-month-old individuals of WT, gcPI4KB F0 crispants were challenged with GCRV-II (5µl/g) by intraperitoneal injection. For gene expression analysis, the fish were maintained in aerated tanks in triplicate with 35 individuals per group with supplemental 35 L water at 25 ± 2 °C. The fish were fed with a commercial pelleted diet at 3 % body weight per day through-out the study. The number of surviving fish was counted daily for 14 days. GraphPad Prism 9.0 was used to generate survival curves, and the log-rank test was used to test differences in survival between the WT, gcPI4KB or gcPI4K2B F0 crispants. The liver, intestine and kidney of 6 WT or gcPI4KB F0 crispants grass carp with or without GCRV-II infection were collected at 5 dpi or 16 dpi, and used for Illumina deep sequencing.