2017 Fiscal Year Research-status 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, 特定研究員 (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 |
Xeroderma pigmentosum (XP) is characterized by defective DNA excision repair. XP patients are vulnerable to UV light (UV) exposure and are prone to develop Basal Cell (BCC) and Squamous Cell Carcinoma (SCC). Somatic mutations in the epidermal stem cells are causative, however, driver genes involved in cancer progression are largely unknown. To identify novel oncogenes and tumour suppresor genes that arise upon UV-induced mutagenesis, I will develop an assay of skin carcinogenesis using XPA patient-derived iPS cells. I have developed a keratinocyte differentiation protocol that after 28 days yields between 20~30% of cells expressing the epidermal markers KRT5 and KRT14. It is possible to isolate epidermal progenitor cells by flow-cytometry and use them to form a 3D-reconstructed epidermis.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Human iPSCs from XPA patients were generated and the mutation located in the intron splice acceptor in exon 4 of XPA, IVS3AS, G-C / IVS3AS, G-C was verified.
A key milestone was achieved by correcting the mutation conferring to XPA, thus generating isogenic iPS cell lines that will serve as reference to compare the XPA genotype within the same cellular background. I will engineer genetically the aforementioned cells to express CRISPR/Cas9 from the AAVS1 locus.
A library of five distinct single-guide RNAs (sgRNA) complementary to the transcription start sites (TSS) of tumor suppressor genes was generated with the aim to knock out their expression with Cas9. The list of target tumor suppressor genes includes: TP53, CDKN1A (P21), CDKN2A (comprising P16-INK4A and P14-ARF), NOTCH and PTEN.
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| Strategy for Future Research Activity |
To elucidate genes/pathways required for the progression of skin cancer, I will use our hiPSCs-skin keratinocyte differentiation and stratification system using XPA-derived hiPSCs or their isogenic control cells followed by UV light-induced mutagenesis to induce BCC and SCC.
1. Use XPA-derived hiPSCs or their isogenic control cells to differentiate and prepare 3D-reconstructed epidermis.
2. Use CRISPR/Cas9 to ablate tumor suppressor genes followed by UV-irradiation to induce mutagenesis.
3. Identify, analyze and validate the mutations that conferred malignancy in the screen.
The strategy outlined above is very likely to generate tumors, however, to encourage transformation in our cancer model, I will generate RASG12D activating mutation into the XPA hiPSCs and their isogenic control cells.
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| Causes of Carryover |
During fiscal year 2017, the experiments performed were focused mainly in molecular biology experiments such as generation of targeting vectors and single guide RNA vector preparation. Many reagents used were already stocked in the laboratory of Dr. Woltjen, a kenkyu-buntansha in this project, who kindly shared them. However, in the current fiscal year 2018, many experiments involving tissue culture of iPSCs and differentiation reagents will be necessary such as cytokines, antibodies and other consumables, therefore carry over of unspent funds is gratefully received.
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