Evaluating the role of cis-regulatory tandem DNA repeats in human disease and evolution
Project/Area Number |
21H02460
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Research Category |
Grant-in-Aid for Scientific Research (B)
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Allocation Type | Single-year Grants |
Section | 一般 |
Review Section |
Basic Section 43050:Genome biology-related
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Research Institution | Kyoto University |
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)
|
Co-Investigator(Kenkyū-buntansha) |
川路 英哉 公益財団法人東京都医学総合研究所, ゲノム医学研究センター, 副センター長 (20525406)
依馬 正次 滋賀医科大学, 動物生命科学研究センター, 教授 (60359578)
井上 詞貴 京都大学, 高等研究院, 特定准教授 (60525369)
|
Project Period (FY) |
2021-04-01 – 2024-03-31
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Project Status |
Granted (Fiscal Year 2023)
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Budget Amount *help |
¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000)
Fiscal Year 2023: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2022: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000)
Fiscal Year 2021: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
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Keywords | DNAリピート / ゲノム編集 / CRISPR-Cas9 / iPS細胞 / レポーターアッセイ / ゲノム解析 / ヒトiPS細胞 |
Outline of Research at the Start |
The expansion of genomic tandem repeats is coincident with the rapid evolutionary trajectory of hominid species. However, apart from a few rare examples, associations between repeat sequences and biological function remain statistical. How extensively have non-coding DNA repeat variants influenced primate brain evolution and human neurological disease? In this study we will develop a variant classification and genome editing framework in primate stem cells for defining the role of tandem repeats in primate gene regulation and their influence on disease and evolution.
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Outline of Annual Research Achievements |
Using our dataset, we aimed to construct a lentivirus-based reporter assay library for evaluating the activity of enhancers and their polymorphic deletion variants. We surmised that construction of VNTRs in a lentirival vector would face limitations of DNA element size and possible instability due to the repetitive nature of the elements. We therefore developed alternative approaches to measure gene-regulatory properties of VNTRs in a high-throughput manner based on epigenome editing and precision gene editing of endogenous VNTRs. For these purposes, we prepared iPS cell lines for CRISPRi and a protocol for controlling VNTR copy number. In revision, we expect MPRA screens to be performed using minimal cCRE elements identified within VNTR sequences.
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Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
To advance the project, we revised the strategy for high-throughput assessment of VNTRs using epigenome editing and precision gene editing approaches. These alternative approaches will allow us to classify and prioritize variants for characterization in primate ESCs or iPSC-derived in vitro models, enabling the ultimate project goal.
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Strategy for Future Research Activity |
VNTR library screens will incorporate alternative perturbation strategies such as CRISPRi to elucidate variant function. In parallel, additional VNTR loci associated with disease will be analyzed by PCR and long-read Nanopore sequencing to identify polymorphisms amongst healthy human iPSC lines from various ethnic backgrounds. Our novel VNTR editing method developed in FY2022 will be used to generate polymorphic iPSC lines. We will also develop methods to visualize VNTR loci. Genomic analyses of mouse, monkey, and human genomes will be used to identify human- and primate-specific loci by genomic conservation. Such loci will be prioritized for gene editing in mouse and monkey primary cells and embryos as well as human iPS cells using our MHcut design software and novel VNTR editing method.
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Report
(2 results)
Research Products
(5 results)