Abstract: To construct the porcine PPARD gene editing vector and detect the gene editing efficiency. In this study, two sgRNA sequences that can target the cutting of PPARD gene were designed according to the structure and sequence characteristics of PPARD gene, and the two PPARD sgRNA expression boxes were connected to a CRISPR/Cas9 vector in tandem. After the constructed plasmids were transfected into PK15 cells, the DNA of each group of cells was extracted. The mutant region was amplified by PCR, and the vector editing efficiency was detected at the DNA level by sequence determination.The total RNA and total protein of each group were extracted, and the gene editing efficiency of recombinant vectors was detected by qRT-PCR and Western blot at the level of gene expression and protein expression. The total mutation rate of the PPARD-2KO group was 45.83% (22/48). Compared with the normal control group, the PPARD mRNA in the PPARD gene editing group was significantly decreased by 84% (P<0.01), and the PPARD protein expression was significantly decreased by 61% (P<0.01). The results show that this study successfully constructed a high-efficiency PPARD gene editing vector by using the improved CRISPR/Cas9 vector system, and can achieve high-efficiency gene editing on cells without drug screening, which will greatly improve the efficiency of subsequent screening of single-cell cloning and promote the functional study of PPARD genes.

Key words: pig, PPARD, CRISPR/Cas9, gene mutation