Abstract: Objective: To construct a VASN knockout AML12 mouse liver cell line using CRISPR/Cas9 technolo-gy combined with the conditional knockout strategy of Cre-Loxp system.Methods: Two recombinant CRISPR/Cas9 vectors were constructed by designing targets upstream and downstream of the second exon of VASN gene and synthesizing gRNA sequences.Donor vector donor DNA with Loxp sequence and side homologous sequence in upstream and downstream of VASN was designed and constructed.The three plasmids were co-transfected into mouse liver cells AML12, and the monoclonal cell line AML12-Loxp with stable Loxp sequence was obtained by PCR and sequencing detection.pALB-Cre plasmid was transfected into AML12-Loxp, and VASN knockout cell lines were screened and established.Results: Two recombinant vectors of pX459-gRNA-VASN-1 and pX459-gRNA-VASN-2 targeting for knockout of VASN gene and one donor vector were successfully constructed.Twelve single cell clones were obtained after transfection with drug sieve, and 5 monoclonal cells were sequenced to ac-curately tap into Loxp sequence upstream and down-stream of VASN gene.The knockin efficiency of ho-mologous recombination was 41.6%.Twenty single cell clones were obtained after transfection with pALB- Cre plasmid, and 12 VASN gene knockout monoclonal cells were obtained by PCR and sequencing detection.The knockout efficiency was 60%.Conclusion: AML12 cells with VASN gene knockout are successfully constructed using CRISPR/Cas9 technology com-bined with Cre-Loxp system, which lays a molecular foundation for the subsequent in vitro study of the function of VASN in liver cells and the realization of the conditional knockout of VASN in animals.