Abstract: In order to construct an immunodeficient pig model with RAG1 gene knockout by microinjection in one step,CRISPR/Cas9technology was used to design and screen out efficient sgRNA for the RAG1 gene of pigs. It was mixed with Cas9 mRNA transcribed in vitro in a4 ∶ 6 ratio and microinjected into the fertilized egg of pig in vitro fertilization,and then the gene-edited fertilized egg was transplanted into the uterus of the recipient sow. After the piglets were born,the skin tissue of the ear margin was collected and the genome was extracted for PCR amplification,T7 endonuclease I enzymatic identification,and identification of the edited genotype by Sanger sequencing. The survival cycle of immunodeficient piglets with gene knockout was monitored in normal environment. Immune organs such as thymus and spleen of dead gene-edited piglets were collected for histological analysis. The results showed that the knockout efficiency of RAG1 gene was screened at the level of porcine fibroblast cell line. The sgRNA sequences for the high efficiency knockout of exon 2 of porcine RAG1 gene were screened. Two RAG1knockout immunodeficient piglets were obtained by microinjection of sgRNA and Cas9 mRNA mixture into the recipient oocyte. The insertion of1 bp into the second exon base sequence of RAG1 gene resulted in frameshift mutation,which destroyed the function of RAG1 gene and resulted in immune system defect of piglets. The survival time of the two immunodeficient piglets born in the ordinary breeding environment was 19 d and33 d,respectively. Pathological sections and tissue morphology tests were performed on the dead piglets. The phenotypes of the immunodeficient pigs with RAG1 gene knock-out showed abnormalities such as athymism and dysplasia of the spleen,and they could not survive in the normal breeding environment. Spleen structure disorder,inflammatory cell infiltration and other immune system abnormalities. These results indicated that RAG1 knockout immunodeficient pig models could be obtained by microinjection with CRISPR/Cas9 technology.