Objective: Based on the protein structure of SALL1, the homology of SALL1 protein between pig and human was analyzed. Bama miniature pig fetal fibroblast cell lines with Sall1 gene knocked-out were developed in order to providing experimental donor cells for obtaining a pig renal development deficiency model via somatic cell nuclear transfer. Methods: Firstly, bioinformatics methods were used to analyze the structural of SALL1 protein between human, pig and mouse. Secondly, sgRNA was designed on the first exon of pig Sall1 gene and connected to PX330 plasmid to construct Sall1 gene knockout target vector. Then, through the initial cells transfection, the targeting efficiency of PX330?sgRNA vector was verified by Sall1 gene sequencing analysis. Finally, the vector was transfected into primary PFFs. The monoclonal cells were obtained through drug screening and their genotypes were identified. Results: Bioinformatics analysis showed that pig SALL1 protein was more similar to human counterparts than mouse. CRISPR/Cas9 expression vector for Sall1 gene targeting was constructed and 33 monoclonal cell lines were obtained, among them 16 Sall1 biallelic mutant cell lines were identified by gene sequencing. Conclusion:Bioinformatics methods confirmed the higher homology of human and porcine SALL1 proteins. Sall1 -/- cell lines were obtained by CRISPR/Cas9 gene editing technology, which will provided research materials for studying the role of Sall1 gene on the development of pig kidney, and lay a foundation for obtaining the porcine renal development deficiency model in the next step.