CRISPR/Cas9 system consisting of target-specific guide RNA and RNA-guided endonuclease is being used for targeted genome editing in many species. In order to establish an effective approach of targeted mutagenesis using the CRISPR/Cas9 system in primary cultured neurons, we constructed HIV-based lentiviral vectors carrying the CRISPR/Cas9 system. In additon to standard integration-proficient lentiviral vectors (IPLV), integration-deficient lentiviral vectors (IDLV) targeted against M₁ or M₄ muscarinic acetylcholine receptor genes and several other targets were prepared and evaluated in this study. Efficacy of these vectors was examined in cultured cell lines, and also analyzed in primary cultured hippocampal and striatal neurons. Surveyor nuclease assays showed the targeted mutation in the cultured cells transduced by the CRISPR vectors. Fragment-length analysis of genomic PCR products also showed that the lentiviral vectors putatively induced frame-shift mutation in the target sequence. IDLV was more suitable for the CRISPR/Cas9 mutagenesis, because the transgenes were rarely integrated into the host genome and their expressions were transient; however, much higher dose of IDLV was needed for significant induction of mutagenesis than dose of IPLV. The lentiviral vectors constructed in this study should be useful in conducting targeted mutagenesis and functional analysis of M₁ and M₄ muscarinic acetylcholine receptors, in cultured stiatal and hippocampal neurons, as well as in developing embryos and brains of living animals.