Effect Of HTMPRSS2 Gene Knock-in On PEDV Susceptibility In Pigs

COVID-19 pneumonia is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2),which is characterized by fever,cough,muscle soreness and loss of smell and taste.Since the first case of COVID-19 pneumonia was discovered in 2019,SARS-COV-2 has swept the world rapidly,posing a great threat to human life and property.SARS-Co V2 binds to the angiotensin converting enzyme 2(ACE2)to invade the host through S protein,during which transmembrane protease serine 2(TMPRSS2)derived from the host is essential for the cleavage of S protein.Nowadays,it is urgent to develop effective vaccines and drugs against COVID-19,and a suitable SARS-Co V-2 susceptible animal model can play an important role in the development and evaluation stage.The mouse model for COVID-19 research has been developed,while there are many drawbacks.Pigs are very similar to human in terms of volume,organ size,anatomical characteristics,genomics and pathology,making them especially suitable as research models of human diseases.In this study,the idea of site-specific integration of human TMPRSS2 gene into porcine ROSA26 gene locus was proposed.Based on the site-specific integration system established for porcine ROSA26 site in the early stage,porcine fetal fibroblasts with hTMPRSS2 gene site-specific integration at ROSA26 site were successfully screened and obtained,with which two F0 generation pigs expressing human TMPRSS2 were successfully prepared by somatic cell nuclear transfer and embryo transfer techniques.In addition,gene editing pig models are often threatened by porcine pathogen infection in the process of feeding,propagation and individual challenge,which hinders the research process of gene editing pigs used for the subsequent development of vaccine and drug.Studies have shown that overexpression of TMPRSS2 can improve the susceptibility of host cells to PEDV.Therefore,based on CRISPR/Cas9 mediated site-specific gene editing strategy,a variety of TMPRSS2 gene editing cells were obtained using PK15 cells as the research object,and the effects of hTMPRSS2 integration at porcine ROSA26 locus on PEDV infection were discussed,which provided data and reference for subsequent breeding,propagation and individual challenge experiments of hTMPRSS2 gene editing pigs.Specific research results were as follows:1.With CRISPR/Cas9 gene editing system,foreign gene hTMPRSS2 was introduced into ROSA26 locus of porcine genome through homologous recombination,and fetal fibroblasts with site-specific integration of hTMPRSS2 gene were screened.Using these fetal fibroblasts as donor cells,F0 generation pigs expressing hTMPRSS2 gene were obtained by somatic cell nuclear transfer and embryo transfer techniques.Different tissues,fibroblasts and renal cortex cells were isolated from positive and wild-type pigs by dissecting them.2.PK15 cells with hTMPRSS2 gene integrated at porcine ROSA26 site were screened by electrotransfection and monoclonal selection.The wild-type and positive PK15 cells were inoculated with PEDV virus in the same proportion.After RNA extraction and reverse transcription,the difference of virus copy number between the two groups was detected by q PCR.The results showed that the insertion of hTMPRSS2 made PK15 cells,which were not susceptible to coronavirus PEDV,acquire stronger infection ability.Then,the sg RNA targeting vector directed to porcine TMPRSS2 gene was designed,and PK15 cells with p TMPRSS2 knockout were screened by CRISPR/Cas9 gene editing system to confirm that TMPRSS2 participated in PEDV infection.The result of cell inoculation showed that the deletion of TMPRSS2 gene led to the decrease of PEDV copy number in PK15 cells compared with wild-type cells.3.Primary renal cortical cells isolated from positive and wild-type pigs were cultured and infected with PEDV at the MOI of 0.01 to further evaluate the effect of integration of hTMPRSS2 at porcine ROSA26 site on porcine virus at cellular level.The results showed that the copies of PEDV in positive porcine renal cortical cells were 100 times higher than those in wild-type cells.