Objective To construct a mouse model of targeted knockout of Fscb/Cabyr dual genes by using the CRISPR/Cas9 system, in order to found a basis for the study of the role of FSCB and CABYR protein complexe in sperm flagellum movement and capacitation. Methods The targeting sites for CRISPR/Cas9 system were designed according to the full-length sequence of Cabyr gene. Two pairs of single-guide RNA (sgRNA) were chemically synthesized, and then inserted into the linearized plasmid pUC57-T7-gRNA to express sgRNA. The Cas9 RNA and sgRNA were transcribed in vitro by T7 RNA polymerase, and the obtained products were mixed in proper proportion and microinjected into the fertilized eggs of C57BL/6J mice to obtain the primary mouse (F0) with Cabyr gene knockout. After the identification of gene mutation by PCR, 2 chimeric females mice and background mice were selected for backcross breeding, and the Cabyr gene knockout F1 heterozygous mice were obtained. Then they were cross bred with the previously established Fscb heterozygous mice, and finally the Fscb/Cabyr (--/--) double gene knockout homozygous mice were obtained. The deletions of Fscb/Cabyr proteins in double gene knockout homozygous mice were detected by Western blotting and immunofluorescence assay. Results Seven positive F1 mice (3 males and 4 females) with deletion of the Cabyr gene fragment and frame shifting were obtained. The homozygous Cabyr knockout mice were obtained by mating the heterozygous male mice with the female mice. Fscb/Cabyr (--/--) double gene knockout homozygous mice were obtained by repeated cross breeding of Fscb and Cabyr heterozygous mice. Western blot analysis indicated that the expression of FSCB and CABYR protein disappeared in the testis and spermatozoa of the male mice. Immunofluorescence assay showed that there was no fluorescence expression of FSCB and CABYR protein in the principle piece of the sperm flagella. Conclusion Fscb/Cabyr (--/--) double gene knockout homozygous mice are obtained by using the CRISPR/Cas9 system and cross breeding, which laid the foundation of animal model for further study on the function of FSCB and CABYR protein complex.
