Objective      To build the targeting vectors to delete the gene of mouse fibrous sheath CABYR-binding protein (fscb)  by using the CRISPR/Cas9 system

and to establish fscb gene knockout mice to study its function in the sperm activation and capacitation.        Methods      The targeting sites were designed according to the full-length sequence of fscb gene

and 3 pairs of single-guide RNA (sgRNA) were chemically synthesized

and inserted into the linearized plasmid pUC57-T7-GDNA. The Cas9  and sgRNAs were transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA mixtures were microinjected into fertilized eggs to generate mice with targeted mutation. The genotypes of homozygous were identified by PCR

DNA sequencing and Western blotting.       Results      The vector expressing sgRNA was successfully built

and the sgRNA and Cas9 mRNA were transcribted in vitro. The active sgRNA and Cas9 mRNA were then injected into the fertilized eggs of mice

and 39 positive mice as the F0 generation were generated. Three select male mice with clear deletion of the fscb gene fragment and with obvious frame shifting were bred with wild-type female mice

and 5 positive F1 mice (2 males and 3 females) were achieved. The heterozygous males mated with females to generate the F2 homozygous mice. Two different genotypes of homozygous were generated. Western blot analysis confirmed that the expression of FSCB protein in the testis and sperm of the homozygous male mice were disappeared.       Conclusion       The fscb gene knockout model is successfully established by using the CRISPR/Cas9 system

which offer a tool for the function of FSCB protein.