Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
Animals ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Oryzias/genetics* ; RNA, Guide/genetics* ; RNA, Transfer/genetics*

Virus infection consists of entry, synthesis of macromolecular components, virus assembly and release. Understanding of the mechanisms underlying each event is necessary for the intervention of virus infection in human healthcare and agriculture. Here we report the visualization of Singapore grouper iridovirus (SGIV) assembly in the medaka haploid embryonic stem (ES) cell line HX1. SGIV is a highly infectious DNA virus that causes a massive loss in marine aquaculture. Ectopic expression of VP88GFP, a fusion between green fluorescent protein and the envelope protein VP088, did not compromise the ES cell properties and susceptibility to SGIV infection. Although VP88GFP disperses evenly in the cytoplasm of non-infected cells, it undergoes aggregation and redistribution in SGIV-infected cells. Real-time visualization revealed multiple key stages of VP88GFP redistribution and the dynamics of viral assembly site (VAS). Specifically, VP88GFP entry into and condensation in the VAS occurred within a 6-h duration, a similar duration was observed also for the release of VP88GFP-containing SGIV out of the cell. Taken together, VP088 is an excellent marker for visualizing the SGIV infection process. Our results provide new insight into macromolecular component recruitment and SGIV assembly.
Animals ; Cell Line ; Embryonic Stem Cells ; metabolism ; pathology ; virology ; Fish Diseases ; genetics ; metabolism ; virology ; Humans ; Iridoviridae ; physiology ; Oryzias ; Viral Proteins ; genetics ; metabolism ; Virus Assembly ; physiology