Gene editing of swine intestinal organoids to explore proviral factors of porcine epidemic diarrhea virus

Abstract

Porcine Epidemic Diarrhea Virus (PEDV) is a highly contagious enteric coronavirus that targets the small intestinal epithelium in swine. PEDV is highly lethal in neonatal piglets and has the potential to be economically devastating for swine producers if an epidemic were to occur. Since there is limited success with vaccinations, gene editing technology can be leveraged as an effective containment strategy of PEDV. Genes identified as potential receptors or proviral factors can be targeted, creating knock-out (KO) models which can be subsequently challenged with PEDV. The Death Receptor 5- like (DR5-like) gene has been previously associated with promoting early viral infection of PEDV. In this study, DR5-like KO fetuses were created, and small intestinal organoids were established from them, with the goal of using them for viral challenge. Phenotypic abnormalities of these KO animals were analyzed and histological analysis showed morphological differences in the villous structure along with a reduction in intestinal stem cell markers. As intestinal organoids (IO) grow in popularity, and are more often used in viral challenges, direct gene editing of IOs to create these KO models has become more appealing. Directly editing the organoid itself can improve timeline efficiency of creating a novel model and prevents the need for sacrificing an animal to isolate intestinal crypts. The aim of this set of experiments was to refine a ribonucleoprotein-based editing approach tailored specifically for pig intestinal organoids. This refined protocol greatly improved editing of intestinal organoids, with maximum editing up to 100 percent per IO.