Objective To establish a method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9,and analyze its off-target effect.Methods The gene editing site for MAD2L1 gene was designed by CHOPCHOP,and the Cas9-MAD2L1 vector was constructed based on the designed editing site.Cas9-MAD2L1 was then transfected into NIH/3T3 cells and screened with puromycin,followed by observing GFP expression using fluorescence microscopy.The genomic DNA from transfected cells was extracted and a partial fragment of MAD2L1 gene was amplified by PCR.T7E1 analysis and Sangger sequencing were used for gene editing and off-target analysis.Results After Cas9-MAD2L1 transfection and puromycin screening,a large number of GFP-expressing cells were observed under the fluorescence microscope.Combined the PCR result with TE71 analysis,the amplified 228 bp PCR products can be digested into 166 bp and 62 bp fragments.The sequencing result showed that the second exon of MAD2L1 gene was successfully edited,and the off-target effect was undetected in our system.Conclusions The method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9 is successfully established,and off-target effect of MAD2L1 gene is not detected.