Abstract: The pathogenic gene of spinal muscular atrophy（SMA） is named the survival of motor neuron（SMN）. In this study, the CRISPR/Cas9 system was used to analyze the efficiency of sgRNA which targeted upon exon 1 of rabbit SMN gene. The cDNA sequence of SMN gene was analyzed by bioinformatics method and the cDNA was cloned by PCR. 4 sgRNAs were designed for exon 1 of SMN gene, which were sgRNA1-1, sgRNA1-2, sgRNA1-3, and sgRNA1-4. The gene knockout vector pX459-sgRNA and RGS-sgRNA were constructed. The fluorescence effect was observed 24 h after co-transfection of the 293 T cell, and the efficiency of sgRNA was analyzed by flow cytometry. The sequence was confirmed by sequencing analysis. The results showed that the conservation of the CDS about the target gene was high, about 80%. The length of the cDNA was 888 bp, and the protein was encoded with 295 amino acids. The fluorescence detection and the flow cyto-metry analysis showed that the cleavage efficiency of sgRNA on exon 1 from high to low was sgRNA1-2（57.1%）, sgRNA1-3（42.0%）, sgRNA1-1（33.5%）, and sgRNA1-4（0.1%）. The CRISPR/Cas9 gene editing technology can be used as an efficient method to analyze sgRNA efficiency and lay a foundation for constructing rabbit SMA disease model by gene targeting.