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Objective To establish effective CRISPR/Cas9 system targeting mouse miR let-7a. Methods Two pairs of dual-sgRNAs sequences were designed according to the coding sequences of two coding sites let-7a-1 and let-7a-2 of mouse let-7a respectively. The targeting potency of four pairs of sgRNA was analyzed through the construction of sgRNA expression vector, cotransfection of N2a cells, drug screening. Positive cells' PCR products sequencing and TA clone sequencing. Results The four pairs of targeting vectors played a role in the cells, and there were base insertions or deletions at the targeting sites, and the Sanger sequencing peaks of RCR products at the drug screening positive cells showed nested peaks at all four targeting sites.The results of TA cloning sequencing showed that the targeting efficiency of the four pairs of dual-sgRNAs targeting sequences designed in this study was 42.10%, 62.50%, 52.63% and 47.37% respectively. Conclusions The results showed that the four pairs of dual-sgRNAs-directed sequences designed in this study deavaged miR let-7a by inducing Cas9 protein effectively and thus generated a mutating effect in mice. Our study laid a foundation for further exploring of let-7a targets and elucidating the mechanism of its' anti-cancer function.