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2019 Fiscal Year Annual Research Report

CRISPR/Cas9 mediated identification of novel gene targets for XPA-associated skin cancer using in vitro reconstituted skin from iPS cells

Research Project

Project/Area Number 17K07165
Research InstitutionKyoto University

Principal Investigator

オセゲラヤネス ファビアン  京都大学, iPS細胞研究所, 特命助教 (80751304)

Co-Investigator(Kenkyū-buntansha) ウォルツェン クヌート  京都大学, iPS細胞研究所, 准教授 (50589489)
Project Period (FY) 2017-04-01 – 2020-03-31
KeywordsHuman iPSCs / CRISPR/Cas9 / Squamous Cell Carcinoma / Skin differentiation / Xeroderma pigmentosum
Outline of Annual Research Achievements

XPA IVS3AS, G-C mutation was repaired using CRISPR/Cas9 and single-stranded oligonucleotides (ssODN), thus generating isogenic control cells. Two different XPA iPSC lines obtained from distinct donors were successfully corrected and the expression of XPA protein was restored.
UVC treatment (1 J.m-2) of normal, XPA and gene-corrected iPSCs generated DNA mutations that were identified using anti-cyclobutane pyrimidine dimers antibodies. Both normal and isogenic control iPSCs but not XPA-derived iPSCs were able to grow and form colonies upon UV-irradiation, thus confirming that DNA repair was restored in XPA-corrected iPSCs. Nevertheless, XPA-derived iPSCs showed persistent colonies when treated with subacute UV-doses. A decreased growth rate was observed in the isogenic control cells that had been exposed to UVC in comparison to normal iPSCs, suggesting the prior accumulation of mutations throughout the lifespan of the donor patients might impair cell survival. We proceeded to create the pathogenic mutation IVIS3AS G>C in normal iPSCs. Importantly, the cell line with de novo-created XPA mutation provide with a neat genomic background that serve as reference when comparing UVC-irradiated genomes obtained from patient and isogenic control iPSCs using NGS.
Experiments using skin keratinocytes differentiated from iPSCs subjected to UV-induced mutagenesis are underway; the genome obtained from the samples will be analyzed by NGS to identify cancer causative mutations.

  • Research Products

    (9 results)

All 2020 2019

All Journal Article (1 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 1 results,  Open Access: 1 results) Presentation (7 results) (of which Int'l Joint Research: 2 results,  Invited: 4 results) Book (1 results)

  • [Journal Article] OVOL1 Influences the Determination and Expansion of iPSC Reprogramming Intermediates.2019

    • Author(s)
      Kagawa, H., R. Shimamoto, S.I. Kim, F. Oceguera-Yanez, T. Yamamoto, T. Schroeder, and K. Woltjen
    • Journal Title

      Stem Cell Reports

      Volume: 12 Pages: 319-332

    • DOI

      10.1016/j.stemcr.2018.12.008

    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] Applications of genetically engineered human iPSCs to understand skin cancer development and pathology.2020

    • Author(s)
      Fabian Oceguera-Yanez
    • Organizer
      Keystone Symposium on Tissue Organoids
    • Int'l Joint Research / Invited
  • [Presentation] Applications of genetically engineered human iPSCs to understand skin cancer development and pathology. Poster presentation2020

    • Author(s)
      Fabian Oceguera-Yanez, Thomas Luc Maurissen, Michiko Nakamura, Tomoko Matsumoto and Knut Woltjen
    • Organizer
      Keystone Symposia on Tissue Organoids
    • Int'l Joint Research
  • [Presentation] Development and application of genome editing and iPSC differentiation to model skin damage and disease2019

    • Author(s)
      Fabian Oceguera-Yanez
    • Organizer
      第4回 京都皮膚基礎研究会
    • Invited
  • [Presentation] Bringing iPSC Technology to the Bedside2019

    • Author(s)
      Fabian Oceguera-Yanez
    • Organizer
      Dior Skincare Summit, Tokyo
    • Invited
  • [Presentation] Introduction to CiRA and iPSC Research2019

    • Author(s)
      Fabian Oceguera-Yanez
    • Organizer
      Capture Totale Japan Event, Tokyo
    • Invited
  • [Presentation] Application of genetically engineered iPSCs to dissect skin cancer pathology. Poster presentation2019

    • Author(s)
      Fabian Oceguera-Yanez, Thomas Luc Maurissen, Michiko Nakamura, Tomoko Matsumoto and Knut Woltjen
    • Organizer
      MBSJ 日本分子生物学会 2019
  • [Presentation] Applications of genetically engineered human iPSCs to understand skin cancer development and pathology. Poster presentation2019

    • Author(s)
      Fabian Oceguera-Yanez, Thomas Luc Maurissen, Michiko Nakamura, Tomoko Matsumoto and Knut Woltjen
    • Organizer
      CiRA International Symposium 2019
  • [Book] ヒトiPS細胞 のAAVS1遺伝子座への遺伝子組込み。2019

    • Author(s)
      Lee, S., 香川晴信, 松本智子, Oceguera-Yanez, F., and Woltjen, K.
    • Total Pages
      386
    • Publisher
      実験医学
    • ISBN
      ISBN 978-4-7581-2244-3

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Published: 2021-01-27  

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