Objective：In order to detect the putative off-target effects of CRISPR/Cas9 system，the knockout mouse model of RNA methyltransferase Nsun5 gene were constructed using wild-type Cas9 and nickase Cas9-D10A，respectively，and performed off-target analysis of F0 mice to compare the specificity of the two strategies. Methods：Paired sgRNAs targeting the third exon of Nsun5 were designed and co-injected with Cas9 mRNA and Cas9-D10A mRNA respectively into mouse one-cell fertilized eggs to generate Nsun5 knockout founders. Off-target effects were analyzed in founder mice injected with CRISPR/Cas9 and Cas9-D10A，respectively. Results：CRISPR/Cas9 and Cas9-D10A were used to establish the Nsun5 gene knockout mouse model successfully. The founder mice obtained from the two variants were subject to off-target analyzation，and no off-target sites were detected. Conclusion：Nsun5 gene knockout models were successfully generated by the both strategies without off-target sites，paving the way for further studying the biological functions of Nsun5.