Advances in gene editing strategies for viral disease resistance in pigs: Knock-out, knock-in, and overexpression approaches

CRISPR-based screening of host factors in veterinary viral infections: from target discovery to host-directed antiviral strategies

This review systematically summarize recent progress in CRISPR/Cas9-based screening studies of major livestock and poultry viruses, including foot-and-mouth disease virus (FMDV), swine enteric coronaviruses, African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), avian leukosis virus (ALV), and other zoonotic pathogens.

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

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.

CRISPR-Cas9: an emerging and precise genome editing system for crops to combat food insecurity

This review summarizes CRISPR principles, highlights technical breakthroughs and crop applications that mitigate biotic and abiotic stresses, and outlines practical challenges and future directions necessary for responsible deployment in agriculture.

Development of attenuated and inactivated Dengue strains using advanced gene editing tools

Isolation, Characterization and Reverse Genetic System Establishment of a Highly Virulent PEDV Strain

Porcine epidemic diarrhea virus (PEDV) G2c variants have recently emerged, posing significant challenges to swine health management. As a major coronavirus affecting the swine industry, PEDV exhibits extensive genetic variability, which has greatly complicated disease control. Current vaccines provide suboptimal protection under field conditions. Therefore, the isolation of recently circulating strains and the establishment of a robust reverse genetics system are critical for advancing the study of emerging variants and facilitating rational vaccine development. In this study, a PEDV field strain designated PEDV-BJ-2023 was isolated from diarrheic piglets in Guizhou, China. Phylogenetic analysis based on the complete genome and spike gene classified PEDV-BJ-2023 within the emerging G2c lineage. To facilitate functional studies, a full-length infectious cDNA clone was constructed using transformation-associated recombination cloning in yeast. Furthermore, an enhanced green fluorescent protein reporter virus was generated via CRISPR/Cas9-assisted homologous recombination by inserting an EGFP-2A cassette upstream of the nucleocapsid gene. The recombinant viruses displayed virion morphology and plaque characteristics similar to those of the parental wild-type PEDV-BJ-2023 strain, although the parental virus exhibited faster replication during the early stage of infection in vitro. In 5-day-old piglets, all three viruses caused severe diarrhea, weight loss, and intestinal lesions; however, recombinant viruses exhibited slightly reduced viral shedding and pathogenicity, with rPEDV-EGFP being the most attenuated. Notably, rPEDV-EGFP maintained stable EGFP expression over eight serial passages. This study establishes a reverse genetics platform for an emerging G2c PEDV strain and provides a stable fluorescent reporter virus, offering valuable tools for visualizing viral infection and investigating virus–host interactions.

Advances in gene transfer technologies: comparing viral and non-viral vectors for therapeutic applications

This review provides a comprehensive overview of the current status of viral and non-viral vector systems for in vivo and ex vivo applications, and key comparisons are made across safety, efficacy, scalability, and immune responses.