We have previously found that integrin α5β1 on dopaminergic neurons plays an important role in the neurite outgrowth on striatal neurons. This finding indicates that integrin α5 (Itga)-overexpressing dopaminergic neurons enhance functional regeneration in transplantation therapy for Parkinson disease. Here, we generated the dopamine transporter (DAT)-Itga heterozygous knock-in mouse embryonic stem cells using the CRISPR)/Cas9 system. These cells can be induced to express Itga gene after dopaminergic differentiation, avoiding the loss of DAT function. The knock-in targeting vector expressing Venus (KI-Ctrl) or Itga followed by Venus (KI-Itga) contained a transgene of 2721 bp or 6470 bp, respectively, which was flanked by the 5'- and 3'- homology arms. Homology arms of approximately 2 kbp were required to obtain heterozygous recombinant clones. Although two KI-Ctrl cloned cells with accurate chromosomal sequence in non-targeted allele were obtained, all KI-Itga cloned cells had indel mutations. By decreasing the amount of Cas9-expressing plasmid and co-transfecting with the rescue vector containing chromosomal sequences, one KI-Itga5 cloned cells with accurate DNA sequence in non-targeted allele was obtained.