Generation Of PEDV Resistant Gene-edited Pigs By CRISPR/Cas9-mediated Knock-in Strategy

The pathogen of porcine epidemic diarrhea is porcine epidemic diarrhea virus,which has the characteristics of fast transmission,wide prevalence and high mortality of piglets.The outbreak and popularity of PED has caused a devastating blow to the pig industry in China and even in the world.At present,the main prevention and treatment methods for this disease are oral/intramuscular injection vaccine and anti-feeding of disease material,etc.,but the effect is still limited.Therefore,it is urgent to develop new virus prevention strategies to deal with PEDV infection.In recent years,with the rapid development of molecular genetics and breeding,scholars at home and abroad have proposed that it is one of the effective means for comprehensive prevention and control of pig diseases to improve the resistance of pig population to viruses by breeding new pig breeds(lines)with strong disease resistance from the genetic nature.RNAi(RNA interference)technology has been proved to have great potential and unique effects in the field of virus prevention and treatment,and has been successfully used in the prevention and treatment of a variety of viruses.In this study,we first analyzed the genomes of different PEDV strain,according to the highly conserved in PEDV genome-wide area was designed and synthesized 8of si RNA,PEDV susceptible cell line-small intestinal epithelial cell line(IPEC-J2)as the research object,by electroporation transfection ways will the si RNA transfection into IPEC-J2,antiviral infection studies,tapping the poison after 72 h results show that compared with the control group and other si RNA,si RNA-3-2 and si RNA-4-2 has better inhibition of proliferation of PEDV effect,The inhibition efficiency is about 20%.We selected si RNA-4-2 located in the S protein gene as our study object.In order to successfully prepare PEDV resistant transgenic pigs,we prepared sh RNA-KI fetal fibroblast positive clones according to the method of sh RNA-KI small intestine epithelial cell cloning,screened the positive clones by the same limit dilution method,and then prepared the cloned pigs by SCNT and embryo transfer.After 114 days of gestation,sh RNA-KI transgenic pigs were identified by PCR and sequencing after birth,and the isolated cells were used to challenge the sh RNA-KI pigs.The results showed that the isolated somatic cells could reduce the proliferation of PEDV.Finally,sh RNA-KI pigs were propagated to the third generation,and si RNA could be stably expressed,and the results of individual challenge proved that sh RNA-KI transgenic pigs could effectively inhibit PEDV proliferation in vivo and in vitro.In summary,we used the CRISPR/Cas9 system to establish an efficient platform to generate site-specific transgenic porcine cells and individuals.In this study,combined with CRISPR/Cas9 technology,sh RNA inhibiting PEDV proliferation was integrated into the p Rosa26 site of pigs to verify the influence of sh RNA on PEDV infection.Secondly,transgenic pigs resistant to PEDV were successfully obtained by SCNT technology.The progeny experiment results of the third generation showed that the growth and reproductive performance of KI pigs were not different from that of the wild type.Both in vivo and in vitro stimulation experiments showed that KI pigs could inhibit PEDV proliferation.In the near future,the combination of sh RNA delivery technologies will provide new treatment ideas for most coronaviruses.