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
| Project/Area Number |
17K07165
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| Research Institution | Kyoto University |
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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)
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| 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)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | Human iPSCs / CRISPR/Cas9 / Squamous Cell Carcinoma / Skin differentiation / Xeroderma pigmentosum |
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| 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.
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