2021 Fiscal Year Annual Research Report
Regulation of gene expression revealed by genome editing of repetitive and polymorphic cis-elements
| Project/Area Number |
21F21382
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| Research Institution | Kyoto University |
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Principal Investigator |
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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| Co-Investigator(Kenkyū-buntansha) |
MARTINEZ GALVEZ GABRIEL 京都大学, iPS細胞研究所, 外国人特別研究員
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| Project Period (FY) |
2021-11-18 – 2023-03-31
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| Keywords | ゲノム編集 / ゲノム解析 / DNAリピート / ヒトiPS細胞 / genome editing / genome analysis / DNA repeat / human iPS cells |
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| Outline of Annual Research Achievements |
The project aims to identify variable number tandem repeats (VNTRs), intersect them with potential regulatory features such as promoters and enhancers, and use this information for functional analysis by gene editing in human iPSCs. The candidate revised the original genome analysis code to match the intended purpose of this grant. Moreover, additional public genome analysis tools such as repeat identification and ENCODE database intersection were integrated into the analysis pipeline, improving the potential biological relevance of the recovered data. Output methods are being developed to filter data for MPRA library design and direct integration into our laboratory’s human iPSC gene editing process using CRISPR. The candidate then used CRISPR for gene editing of a repeat region which is a candidate for regulation of the ADGRG1 gene. Gene-edited iPSC clones were isolated and identified to have a reduced repeat number by DNA sequencing. These fundamental achievements will enable the subsequent goals of the project.
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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
Goals for this period included establishing a genome analysis pipeline and demonstration of the pipeline in designing and executing gene editing in human iPSCs. The original genome analysis code developed by the candidate required modification to be applied for the intended purpose of this grant. This challenge was met by the candidate and overcome. Then, the candidate was able to concretely demonstrate application of the computational designs with efficient gene editing of a repeat region in human iPSCs. These two major goals were expected to be completed within the period and both were achieved.
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| Strategy for Future Research Activity |
The custom VNTR library will be completed and screened in FY2022. The screen uses a massively parallel reporter assay (MPRA) to test the activity of cCREs predicted to be affected by changes in VNTR repeat numbers in iPSCs or differentiated neurons. Next generation sequencing will be used in each cell type to measure the relative amount of barcoded reporter RNA versus viral DNA copy number to quantify enhancer activity in the assay. From the results, VNTRs found to affect enhancer activity will be associated with neighboring endogenous genes and changes in their expression patterns will be predicted. In parallel, CRISPR gene editing will be performed in human iPS cells to evaluate VNTR enhancer activity in an endogenous genomic context, using differentiation and RNAseq. We plan to focus on VNTRs associated with neuropsychiatric disorders such as schizophrenia and Parkinson's disease, which affect more than 3% of the population worldwide.
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