Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system have been found in some kind of bacteria’s defense system like a mammalian acquired immune system. This system develops as powerful tools to modify the genome in many species including mammalian. In this system, gRNA recruits hCAS9 nuclease to the specific genomic locus according to the base-pairing rule and then, introduces double strand breaks (DSBs). After the DSBs, non-homologous end-joining is occurred and causes small insertion or deletion at the specific site. Thus, in order to rapidly obtain gene-deficient mouse in several genes involved in immune responses, we designed over 10 gRNAs and validated their working with hCas9 expression in cultured cells. After checking, we injected two plasmids expressing hCas9 and single gRNA, which is targeted at an inflammatory cytokine gene, into the pronucleus of mouse zygote and transferred with pseudopregnant mice. Finally, we succeeded in getting several lines of the gene-deficient mouse.