Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression

Abstract

The porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene is mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets that were challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs. Four live founders generated and proven to be biallelic KO, lacking detectable N-glycolylneuraminic acid (NGNA). The founders were bred, and homozygous offspring were obtained. Two-day-old (in exps. I, n = 6, and III, n = 15) and 3-day-old (in exp. II, n = 9) KO and wild-type (WT, same ages in respective exps.) piglets were inoculated with TCID 50 1x10 3 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sow’s colostrum (in exp. III) every 4 hours. In exp. III, the colostrum was offered 6 times and was then replaced with Ringer/5% glucose solution. At 72 hours post-PEDV inoculation (hpi), the animals either deceased or euthanized were necropsied and intestines were sampled. In all 3 experiments, the piglets showed apparent outward clinical manifestations suggesting that infection occurred despite the CMAH KO. In exp. I, all 6 WT piglets and only 1 of 6 KO piglets died at 72 hpi. Histopathology and immunofluorescence staining showed that the villus epithelial cells of WT piglets were severely exfoliated, but only moderate exfoliation and enterocyte vacuolization was observed in KO piglets. In exp. II, delayed clinical symptoms appeared, yet the immunofluorescence staining/histopathologic inspection (I/H) scores of the two groups differed little. In exp. III, the animals exhibited clinical and pathological signs after inoculation similar to those in exp. II. These results suggest that porcine CMAH KO with nullified NGNA expression are not immune to PEDV but that this KO may lessen the severity of the infection and delay its occurrence.

The Role of Host Genetic Factors in Coronavirus Susceptibility: Review of Animal and Systematic Review of Human Literature

“… 55 In a similar vein, knockout of CMAH (hypothesized to affect cellular binding) does not result in immunity to PEDV but appears to improve outcomes. 56 …”

Improvements in Gene Editing Technology Boost Its Applications in Livestock

“…More recently, farm animals with point mutations and gene insertions (KI) have been successfully produced using ssODN donor sequences, CRISPR/Cas9 base editing and CRISPR/Cas9 nickase approaches ( Table 3 ). The applications of gene editing technologies for generation of livestock are very diverse, ranging from enhancing important production traits such as meat, milk, and fiber production ( Deng et al, 2014 ; Crispo et al, 2015 ; Hu et al, 2017 ; Zhou et al, 2017 ; Zhang R. et al, 2020 ) to improving disease resistance, health, reproductive efficiency, facilitating animal welfare, and developing new biomedical models to better understand the etiology of diseases and develop novel mechanism-based therapeutic approaches ( Vilarino et al, 2017 ; Fan et al, 2018a , b ; Tu et al, 2019 ; Tanihara et al, 2020 ).…”

Current status of the application of gene editing in pigs

“…(2019) [ 150 ] CMI ANPEP Cas9 mRNA and gRNA 13/18 (72.2) 3/13 (23.1) Tu et al . (2019) [ 151 ] CMI CMP-N-glycolylneuraminic acid hydroxylase Cas9 mRNA and gRNA 5/6 (83.3) 3/5 (60.0) Chen et al . (2019) [ 144 ] CMI TYR, IL2RG, RAG1 Cas9 mRNA and gRNA 15/16 (93.8) 5/15 (33.3) Tanihara et al .…”