Application Of OPTI-CGBE In Swine Disease Resistance Gene Editing

How to prevent infectious diseases in pigs has always been a difficult problem for people to overcome,and swine disease is the biggest threat facing the pork industry.In the past few years alone,hundreds of billions of dollars have been lost globally to various swine diseases,most of which are caused by viruses.These viruses not only endanger the pig breeding industry,but also may endanger human health,how to prevent and rectify pig infectious diseases has always plagued farmers.Unfortunately,as of now,there are no safe and reliable ways to prevent or control disease in these pigs,and economic and animal losses can only be reduced by breeding disease-resistant pigs.Traditional methods of breeding pigs with specific traits is a slow process that can take many years.But with the advent of gene editing technology,it can quickly produce genetically modified F1 generations,which brings hope to the direction of pig breeding.At present,researchers have used the CRISPR/Cas system or single-base editor CBE and ABE to target pig disease resistance genes to breed disease-resistant pig breeds.At present,there are no relevant reports of the application of the CGBE system to pig disease resistance breeding.The CGBE system optimization of the single-base editor in the early stage of the laboratory obtained highly specific OPTI-CGBEs,which improved the editing efficiency of C To G and reduced the risk of off-target.According to the editing window and motif preference of the highly specific OPTI-CGBE,appropriate sg RNAs were designed and introduced into the stop codons of the pig disease resistance-related genes CD163,P53,RIGI,and RNase L in advance,and the related gene knockout monoclonal cells were established.In order to evaluate the safety of the sg RNA,Cas-Offinder predicts possible off-target sites to analyze whether there is sg RNA-dependent off-target,and the transcriptome level analyzes whether there is random off-target,to lay a certain foundation for breeding disease-resistant pig breeds.The results of the study are as follows:(1)Verification of editing efficiency of highly specific OPTI-CGBE on porcine PK-15 cells: A total of 32 sg RNAs were designed for the four porcine CD163,P53,RIGI,and RNase L genes,and co-transfected with OPTI-CGBE into pigs In PK-15 cells,sequencing results showed that CD163 sg RNA2(7.83%),P53sg RNA1(2.15%),RIGIsg RNA1(19.33%),and RNase Lsg RNA2(6.84%)had prematurely introduced stop codons on related genes.It shows that the use of OPTI-CGBE can target the four genes of CD163,P53,RIGI and RNase L in pigs to introduce stop codons in advance.(2)Identification of the safety of the screened sg RNAs: Analysis of monoclonal cell lines constructed by editable sg RNA RNA-seq found that the number of RNA SNVs in CD163 sg RNA1 and P53 sg RNA2 monoclonal cell lines was not the same as the number of SNVs in negative cells PK-15 Significant difference(number of SNVs generated by random RNA off-targets,CD163 sg RNA1:P53sg RNA2:PK-15=38050:40185:43813),and no mutation bias,also proved that the monoclonal cell lines CD163 sg RNA1 and P53 sg RNA2 can reduce random RNA off-targets;The monitoring analysis of possible off-target sites predicted by Cas-OFFinder found that none of the 10 possible off-target sites of CD163 sg RNA2 and the 9 possible off-target sites of P53 sg RNA1 had random off-target problems.(3)Detection of proliferation ability and cell viability of monoclonal cell lines: By studying the cell proliferation experiments of monoclonal cell lines CD163 sg RNA2,P53 sg RNA1 and wild-type PK-15,the results showed that there were no significant differences in cell proliferation ability and cell viability between monoclonal cell lines CD163 sg RNA2,P53 sg RNA1 and wild-type PK-15.In summary:In this study,the highly specific OPTI-CGBE system was used to successfully introduce stop codons in 4 pig disease-related genes in advance,screened sg RNAs with high C To G editing efficiency,and constructed mutant cell lines.Through transcriptome sequencing and sg RNA-dependent Analysis of off-target sites showed that OPTI-CGBE did not cause transcriptome-and sg RNA-dependent off-targets,and had no significant effect on cell proliferation and growth.This study proves that the highly specific OPTI-CGBE system is safe and feasible in pig cells,and provides a basis for subsequent researchers to use single base editors to study and prepare disease-resistant pig breeds in pigs.