Generation Of CD163 Knock Out Pigs And Identification And Differential Analysis Of Regulatory Elements In Different Sensitive Cells To PRRSV

China is the biggest country in pig industry in traditional and sustain the biggest pig population and pork production in the world.However,one of the serious problems in this filed is how to control the constantly emerging various infectious diseases which leads to tremendous economic losses annually.Long-term spread of these infectious viruses among pigs will lead to the variation in the viruses,especially for RNA viruses.The vaccines and veterinary drug used in traditional also couldn't solve this problem and drug abuse and overdoses is harmful for pigs.Instead,with the development of technology in molecular biology,genetic modification of key factors for virus infection in host genome,is more and more popular in this filed.Nonetheless,it's still a challenge work for researchers to discover the candidate factors involved in disease resistance accurately and rapidly.CRISPR/Cas9 system,the third generation of genome editing technology,has been adapted to almost all aspects in the life science.In particularly,the CRISPR/Cas9 technology help three scientists to win the Nobel Prize for Chemistry last year which will make this technology a hit in molecular biology as well as agricultural animal genetics,breeding and reproduction.What's more,with the rapid development of 3D genome technology,it's convenient to identify regular element in cellular level;In this study,we produced the CD163 gene SRCR5 domain-deleted clone pigs via CRISPR/Cas9 system;And then,we identify an alternative “safe harbor” locus in the pig genome.Subsequently,we knocked the CD163 gene coding sequence into Rosa26 locus in 3D4/21 cell lines,and the results indicate that 3D4/21-CD163 cell line is sensitive to Porcine reproductive and respiratory syndrome virus(PRRSV);Finally,we identified the regular element in 3D4/21 and 3D4/21-CD163 cell lines in the help of RNA-seq and CUT&Tag technology.And the detailed results are as follows:(1)Established the experiment platform for porcine fetal fibroblast(PFF)isolated and culture by comparing three methods.And then,we produced the CD163 gene SRCR5 domain-deleted clone pigs via CRISPR/Cas9 system and somatic cell nuclear transfer(SCNT).(2)We provide a new strategy for efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination.This strategy has no influence on the expression of the endogenous GAPDH gene.Thus,the GAPDH locus is a new alternative safe harbor locus in the pig genome for foreign gene knockins.(3)In order to produce PRRSV-sensitive cell lines,the porcine CD163 gene coding sequence(CDS)was knocked in the Rosa26 locus in 3D4/21 cell lines via CRISPR/Cas9-mediate homologous recombination.And the subsequent experiments show that the 3D4/21-cell lines can expression CD163 protein and it's sensitive to PRRSV.(4)From the analysis of RNA-seq data of 3D4/21 and 3D4/21-CD163 cell lines,we identified 1199 differentially expressed genes(DEGs).Among them,374 genes were up-regulated expression and 825 genes were down-regulated expression.And the Gene Ontology(GO)analysis showed that,a lot of DEGs are clustered in cell adhesion,intracellular signal transduction,signal transduction and cell surface receptor signaling pathway.(5)The analysis of differentially expressed genes(DEGs)by RNA-seq and CUT&Tag data,H3K4me3 and H3K27 ac signal were significant enriched in up-regulated genes which indicate that integration of CD163 gene in 3D4/21 cell lines can activate some regulate elements,thus causing 3D4/21 cell more active than before.(6)The enrichment of CTCF peaks at promoters in 3D4/21 and 3D4/21-CD163 cells were significantly correlated with gene expression changes;And loss of CTCF peaks at immunogens in 3D4/21-CD163 cells may leads to down-regulation those genes.(7)Identification of loop numbers at 3D4/21 and 3D4/21-CD163 cells were 6292 and 10981,respectively;Among these loops,the numbers of promoter-promoter interaction loop were 362 and 1662;the numbers of promoter-enhancer interaction loop were 434 and 1283;Further analysis indicate that contact distance of those loop in3D4/21 cells were mainly enriched in short-range interaction and 3D4/21-CD163 cells were mainly enriched in short-range interaction;...