Preparation Of An Immunodeficiency Pig Model Of RAG1 Knock-out Using Gene Editing Tools

Recombinant activating genes（Rags）contain RAG1 and RAG2,which are two similar genes on the autosomal body.The enzymes encoded by them can form protein complexes and play a key role in the V（D）J rearrangement process.The RAG1 gene can encode enzymes that catalyze the recognition of immunoglobulin（Ig G）or T cell receptor（TCR）by T/B lymphocyte precursors during maturation,and bind to the recombinant signal sequence in the gene fragment to initiate V（D）J recombination to produce the diversity of the primary immune library of T/B cells.If this gene is knocked out,V（D）J recombination will be damaged,and the development of T/B cells will stagnate in an immature state,resulting in the inability to form functional T/B lymphocytes,in order to achieve the effect of immunodeficiency.Since pigs are more similar to humans than rodents in terms of physiological and biochemical indicators and organ size,severe combined immunodeficiency model pigs with genetic mutations can be used as a valuable tool for studying the pig immune system.It is also an important model animal for human medical research.RAG1 is necessary for the maturation of pig T/B lymphocytes.After RAG1 is knocked out,model pigs exhibit a severe combined immunodeficiency phenotype with T/B cell defects similar to rodents and humans.In this study,the RAG1 gene was selected as the target gene,and a total of 5 sg RNAs were designed on the exon of the pig RAG1 gene,namely sg RNA-RAG1-1～5,and then the targeting efficiency of 5 sg RNAs was screened by cell transfection.Among them,the efficiency of sg RNA-RAG1-2 is up to 60%.After in vitro transcription is carried out together with Cas9,Cas9-m RNA and sg RNA are directly injected into the cytoplasm of the in vitro fertilized embryo,and placed in the embryo culture medium to observe the development of the embryo.The injected embryos were transplanted into three recipient sows.30 days after the transplantation,medical imaging confirmed that there was a pregnant sow,and the pregnant sow was transferred to the delivery room to be delivered.In the end,3 piglets were born,and then the ear margin tissue of the raw piglets was removed for genotyping,and the spleen and other tissues were taken for backup after the piglets died.The results showed that after genotyping,there were two RAG1^-/-fetuses in the piglets born.At the same time,after H&E staining,it was found that in the spleen of RAG1^-/-born pigs,the lymphatic vessels around the central artery were stunted,and the white marrow almost disappeared.On the other hand,in this study,the RAG1 gene was knocked out by the CRISPR/Cas12i system in Bama pig fibroblasts.Nine sg RNA sequences were designed on the first exon of the RAG1 gene and sg RNA efficiency screening was carried out,of which the knockout efficiency of sg RNA-RAG1-2 was 92.9%.Then Cas12i and sg RNA-RAG1-2 were co-transfected into fibroblasts through nuclear electrotransfection to construct RAG1^-/-monoclonal cell lines.The genotype of the monoclonal cell line was identified,and a131bp knocked out RAG1-15 monoclonal cell line was obtained,and the cell line was used as the donor cell for somatic cell nuclear transplantation,the development of the cloned embryo was observed and the blastocyst was collected for identification.The blastocyst identification results showed that the blastocysts obtained were all embryos with RAG1 gene 131 bp knocked out.In summary,this study produced a RAG1 gene knockout pig model by injecting Cas9m RNA and sg RNA directly into the embryonic cytoplasm of in vitro fertilization.It aims to genetically modify and produce RAG1 pig strains through microinjection of in vitro fertilized embryos to meet the needs of biomedical research.At the same time,this study used the CRISPR/Cas12i system to construct a pig fibroblast line with RAG1 gene knock-out,and obtained RAG1 knock-out blastocysts through somatic cell nuclear transplantation technology.Offspring can obtain RAG1^-/-model pigs without mating and breeding,which greatly shortens the breeding cycle.The significance of this research lies in the use of microinjection technology to obtain RAG1^-/-gene knock-out SCID model pigs in a direct one-step method.At the same time,the CRISPR/Cas12i gene editing system with independent intellectual property rights in China is also used to construct RAG1 gene knock-out pig fibroblasts for future large-scale preparation of RAG1^-/-model pigs through somatic nuclear transplantation technology.RAG1^-/-model pigs can be used in preclinical biomedical research and bridge the gap between small animals and humans.The injection of fertilized eggs by the CRISPR/Cas9 system can efficiently prepare genome-modified pigs,thereby establishing large animal models of important human diseases to bridge the gap between small animals and humans,and opening up unlimited possibilities for the application of livestock genome engineering in agriculture and biomedicine.