Objective：The concept of animal organs regeneration via stem cells injection and blastocyst complementation has been proved. To apply this principle for organ regeneration，the specific organ-deficient large animals must be created firstly in order to make the specific organ ‘developmental niche’. In this study，we attempt to generate the Six1-deficient and Six1/Six4-deficient pig cell lines by CRISPR/Cas9，in order to construct pig kidney-deficient model next step. Methods：The first exon of Six1 and Six4 gene was chosen for the target to design the sgRNA and the two sgRNAs were cloned into PX330 plasmids containing Cas9 skeleton respectively. The efficiency of PX330-sgRNA vector was tested by T7EN1 enzyme assay analysis，then，the efficient PX330-sgRNA was co-transfected with the G418 resistant plasmid（pCMV-tdTomato）into the primary porcine fetal fibroblasts（PFFs）. The monoclonal cell populations were obtained through the G418 screening and the genotypes of monoclonal cells were identified by sequencing analysis. Results：Cas9/sgRNA expression vectors for Six1 and Six4 gene targeting were constructed successfully. Total 48 monoclonal cell populations with presumed Six1 gene mutation were obtained and identified by PCR，among them 21 cell colonies showed Six1 bialellic mutation（Six1-/）. By the same way，we obtained total 44 monoclonal cell populations for both Six1 and Six4 targeting and 13 of them showed Six1-/-Six4-/- genotypes. Conclusion：The Six1-/- and Six1-/- Six4-/- cell lines were obtained by highly efficient CRISPR/Cas9 targeting，which can provide the possibility in production of the pig kidney-deficient model and also be helpful for the function investigation of Six1 and Six4 genes in pig kidney development.