Objective:To establish Vasohibin-2(Vash2) knockout mouse model with CRISPR/Cas9 gene targeting technology. Methods:A selected gene sequence of Vash2 was amplified with the primers of single-guide RNA (sgRNA),and then cloned into the plasmid pUC57-T7-GDNA. The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro. Transcribed gRNA/Cas9 mRNA was microinjected into the mouse zygote. The frame shifting mutation was validated by PCR and gene sequencing. Both the (F0 and F1 generation) knockout mice were analyzed. Results:The vector expressing sgRNA were successfully built. sgRNA and Cas9 mRNA were successfully transcribed and microinjected into mouse zygote. Five positive mice as the F0 generation were identified by gene sequencing. The No.5 mouse was selected to mate with wild-type mice,then achieved F1 generation were mated and produced F2 generation. The frame-shifted of Vash2 knockout mice (F2 generation) were evaluated by PCR and mutations were stably transmitted to the next generation. Conclusion:The Vash2 knockout mouse model was successfully built by Cas9/RNAsystemgene targeting technology,and it could be an efficient tool for Vash2 study.