Generation Of CD163 Modified Pigs And ?-Gal/Neu5Gc/Sd~a Delete Pigs With Gene-Editing Technique

Porcine reproductive and respiratory syndrome?PRRS?caused by porcine reproductive and respiratory syndrome virus?PRRSV?is a worldwide infectious disease which characterized by low reproductive capacity of sows,accompanied by respiratory disorders,slow growth of piglets,high morbidity and high mortality.At present,the main way to prevent and control PRRSV is vaccine immunization.However,due to the immune escape mechanism and antibody-dependent enhancement effect of the virus,the effect of vaccine immunization is not significant.Current results indicate that porcine CD163 is a key receptor for PRRSV infection in host cells recently.The fifth domain of CD163 protein?SRCR5?is a key domain that mediates the interaction between PRRSV and host cells.It binds to viral GP2a and GP4 protein dimers during viral infection,thereby mediating PRRSV invasion of host cells.Therefore,SRCR5 is a target gene manipulation for the preparation of anti-PRRSV cloned pigs.At the same time,pigs have highly homologous gene sequences compared with humans,and is similar to human in organ size and function,anatomical characteristics,physiological functions,etc.Therefore,it is currently considered as the most ideal donor for xenotransplantation to solve the shortage of human organs.In the clinical application process,the transplantation of xeno-transplants from pigs to non-human primates will cause severe super-immune rejection,which will cause the donor organs to lose function in a short time,resulting in transplant failure.At present,the surface antigens of pig-derived cells that can cause super immune rejection include?-Gal??-1,3 galactose?,Neu5Gc?N-acetylneuraminic acid?and blood group antigen Sd^aIn recent years,the rapid development of CRISPR/Cas technology and base editing technology has been successfully applied to a variety of animals and plants,including mice,zebrafish,rabbits,pigs,sheep,etc.showing a great application value in animal disease breeding and xenotransplantation.The purpose of this study was to prepare PRRSV-resistant CD163 modified pigs by CRISPR/Cas9 technology,to explore the applicability of be4-gam system in pig gene editing,and use the BE4-Gam system to prepare?-Gal/Neu5Gc/Sd^a-deficient pigs for future to provide new thoughts on constructing xenotransplantation antigen-deficient pigs.Firstly,this experiment successfully replace the pig CD163 SRCR5 gene sequence with the corresponding gene sequence of pig CD163 SRCR3 using the CRISPR/Cas9system to prepare CD163^SRCR3 pig.At the same time,CD163^E7D pig with CD163SRCR5 knockout was also prepared using CRISPR/Cas9 system.The results of Hp ELISA showed that under normal physiological conditions,the free Hp content in serum of CD163^SRCR3 pigs was not significantly different from that of wild-type pigs.The results of hb-hp complex uptakion showed that under normal physiological conditions,the scavenging ability on hb-hp of macrophages?PMMs?derived from peripheral blood mononuclear cells?PBMCs?of CD163^SRCR3 and CD163^E7D was not significantly different from that of wild-type pigs.Finally,we isolated alveolar macrophages?PAMs?derived from CD163SRCR3 pigs,CD163^E7D pigs and wild-type pigs for infection of HP-PRRSV in vitro.After 72 hours,the results of Q-PCR showed that the PAMs from both CD163^SRCR3 and CD163^E7D pigs could effectively inhibit the proliferation of HP-PRRSV in vitro,and compared with the CD163^E7D,the PAMs from CD163^SRCR3 pig had a more significant inhibitory effect on HP-PRRSV.Subsequently,a corresponding sg RNA was designed for pig?-Gal?Neu5Gc?Sd^a antigen synthesis genes GGTA,CMAH and B4galNT2,respectively,to explore the applicability of be4-gam system in pig cells.The results showed that the single gene editing efficiency of BE4-Gam system achieved 5.3%-10.1%and 66.7%-71.4%in PFFs and parthenogenetic blastocysts respectively at GGTA1?B4GalNT2?CMAH loci.At the same time,the triple gene editing efficiency of BE4-Gam achieved 7.5%and 18.1%respectively.Further studies showed that the AncBE4max system could achieve a nearly 5-fold improvement in single-gene editing efficiency at the target gene sites of parthenoblastocyst and fetal fibroblasts,and a 71.4%improvement in 3-gene editing efficiency at the embryo level when compared with the be4-gam system.Finally,F0 generation deletion pigs with GGTA1,B4GalNT2 and CMAH gene knockout were obtained by BE4-gam system,SCNT and IVF technology.The off-target detection results showed that no off-target mutation was found at the potential off-target sites of F0 generation cloned pigs.The results of IFA antigen test also showed that we successfully prepared piglets with simultaneous deletion of?-Gal,Neu5Gc and Sd^a.In summary,this study successfully prepared anti-HP-PRRSV CD163^SRCR3 pigs,CD163^E7D pigs and?-Gal/Neu5Gc/Sda antigen-deficient pigs using CRISPR/Cas9system and BE4-Gam system,respectively,which provided a good foundation for maintaining the stability of the pig industry and the development of biomedicine.At the same time,this experiment also confirmed that the applicability of be4-gam in pig cells will provide efficient technical means for the construction of xenotransplantation pig,human disease model and more accurate animal character improvement in the future.