Therapeutic antibodies have become an important focus of the biopharmaceutical industry. The Chinese hamster ovary (CHO) cell line is a major host for therapeutic antibody production. To construct productive CHO cell lines, two major transfection methods are commonly used, i.e., random integration and gene targeting. Random integration is a common method in which randomly integrated transgenes cause variation in antibody productivity because they are located in various chromosomal regions that affect transgene expression levels. Recently, gene-targeting methods, in which exogenous genes are inserted into a specific chromosomal region, have improved remarkably. Gene targeting is based on homologous recombination using sequences targeting a specific genomic region of the host cell. Homologous sequences located on both sides of the exogenous gene are used. We used the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system as a gene-targeting method. The CRISPR-Cas9 system induces double-strand breaks (DSBs) via guide RNA and Cas9, which increases the efficiency of homologous recombination . Guide RNA hybridizes to a target integration site and induces Cas9 protein expression, leading to DSB. Finally, the Cas9 protein cuts genomic DNA. In this study, we constructed a simple gene-targeting method in CHO cells using the CRISPR-Cas9 system in which CRISPR vectors induce DSBs and gene-targeting vectors are inserted at the DSB site. In the conventional method, gene-targeting vectors should contain homology arms for effective recombination. In this study, we used the CRISPER system without homology arms for gene-targeted recombination.