Successful correction of factor V deficiency of patient-derived iPSCs by CRISPR/Cas9-mediated gene editing

Takayuki Nakamura, Satoshi Morishige, Hidetoshi Ozawa, Kenji Kuboyama, Yoshitaka Yamasaki, Shuki Oya, Maki Yamaguchi, Kazutoshi Aoyama, Ritsuko Seki, Fumihiko Mouri, Koichi Osaki, Takashi Okamura, Shinichi Mizuno, Koji Nagafuji

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)


Background: Factor V (FV) deficiency is a monogenic inherited coagulation disorder considered to be an ideal indication for gene therapy. To investigate the possibility of therapeutic application of genome editing, we generated induced pluripotent stem cells (iPSCs) from a FV-deficient patient and repaired the mutation of factor V gene (F5) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9). Methods: The patient's peripheral blood mononuclear cells were reprogrammed for iPSCs. The targeting vector was designed with homology arms against F5 containing the corrected sequence. Cas9 ribonucleoprotein (RNP) complex and targeting vector were electroporated into iPSCs. Gene-edited iPSCs were differentiated into hepatocyte-like cells (HLCs). Results: The mutation of F5 in patient-derived iPSCs was repaired by CRISPR/Cas9. In concentrated culture supernatants of patient-derived iPS-HLCs, neither FV antigen nor activity was detected, while in those of gene-corrected iPS-HLCs, FV antigen and specific activity were 67.0 ± 13.1 ng/mL and 173.2 ± 41.1 U/mg, respectively. Conclusions: We successfully repaired the mutation of F5 using the CRISPR/Cas9 and confirmed the recovery of FV activity with gene-corrected iPS-HLCs. Gene-edited iPSCs are promising for elucidating the pathophysiology as well as for a modality of gene therapy.

Original languageEnglish
Pages (from-to)826-833
Number of pages8
Issue number5
Publication statusPublished - Sept 1 2020

All Science Journal Classification (ASJC) codes

  • Hematology
  • Genetics(clinical)


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