2018 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
|
|---|
| Research Institution | Kyoto 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, 特定研究員 (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
|
| Keywords | Human iPSCs / CRISPR/Cas9 / Squamous Cell Carcinoma / Skin differentiation / Xeroderma pigmentosum |
|---|
| Outline of Annual Research Achievements |
Xeroderma pigmentosum (XP) is an autosomal recessive disorder caused by mutations in genes involved in the nucleotide excision repair pathway (NER). Patients defective in NER are prone to develop skin malignancies due to abnormal sensitivity to sunlight. XP-associated skin cancer is a prototypical model of cancer caused by somatic mutations in the epidermal stem cells, however, driver genes involved in skin cancer progression remain largely unknown. To identify novel oncogenes and tumour suppresor genes that arise upon UV-induced mutagenesis, I aim to develop an assay of skin carcinogenesis using XPA patient-derived iPS cells. Firstly, by differentiating XPA-patient iPSCs or their isogenic control cells into skin keratinocytes; and secondly, by setting up a step-wise mutational approach aided by UV-induced mutagenesis and CRISPR/Cas9 to induce cutaneous squamous cell carcinoma (cSCC)in vitro.
XPA iPSCs were derived from XP patients that harbor a mutation located in the splice acceptor of intron 3 of XPA (IVS3AS, G-C / IVS3AS, G-C) (Satokata et al., P.N.A.S. USA. 1990). I applied CRISPR/Cas9 and ssODN to repair the mutation and generated isogenic genetically-corrected cells. The control cell lines will provide with a genomic mutational reference background. Furthermore, I engineered genetically the aforementioned cells to express Cas9 nuclease from the AAVS1 locus following the methodology described previously (Oceguera-Yanez et al., Methods. 2016). Transfection of vectors encoding for sgRNA into Cas9-expressing cells will be performed to knock out tumor suppressor genes.
|
| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
I engineered genetically XPA-patient derived iPSCs and their isogenic control cells to express Cas9 from the AAVS1 locus using the methodology described in our previous report (Oceguera-Yanez et al., Methods. 2016). However, it took a great effort to establish XPA iPS cell lines correctly targeted due to the high avidity of CRISPR/Cas9 system to cleave the AAVS1 loci at both alleles. Often were detected indels at the “non-targeted” WT allele. To overcome this problem, TALENs were used instead. Thus it was necessary to increase the number of targeting experiments and genotyping analysis causing a delay in the schedule. The established cell lines will be used to generate knock out of tumor suppressor genes.
The response to UV irradiation in the aforementioned cells was tested. Preliminary results showed that DNA adducts are generated in iPSCs treated with 1 J m-2 UVC. Currently we are setting up conditions for UV-B irradiation of stratified epidermis.
|
| Strategy for Future Research Activity |
XPA-patient derived iPSCs and their isogenic control cells expressing Cas9 constitutively will be differentiated to keratinocytes. I will expose the iPSCs differentiated to keratinocytes to UV light-induced mutagenesis. Additionally, tumor suppressor genes will be knocked out using Cas9 guided by chimeric RNAs. We have previously prepared vectors suitable for expression of sgRNAs complementary to the transcription start sites (TSS) of the following tumor suppressor genes: TP53, CDKN1A (P21), CDKN2A, NOTCH and PTEN. In the event that no cell transformation is observed, I will generate RASG12D activating mutation to encourage transformation in our cancer model. The mutations that conferred malignancy in the screen will be analyzed and those with UV-light-induced signature will be selected for future study.
|
| Causes of Carryover |
In FY 2018, experiments involving tissue culture, gene editing and genotyping of iPSCs were carried out. To characterize UVC-induced DNA damage in iPSCs, antibodies and ELISA kits were purchased.
During FY 2019, a majority of experiments to differentiate iPSCs will be performed and reagents such as cytokines, antibodies and tissue culture consumables will be required.
Reagents to isolate total Nucleic Acid such as DNA and RNA and kits to generate libraries suitable for massive parallel sequencing of DNA using NGS will be required in order to analyze UV-induced mutagenesis in keratinocytes. Additional costs for the preparation of histologic specimens through an outsource company are calculated too. Therefore carry over of unspent funds will be gratefully received.
|
Research Products
(2 results)
