| 研究課題/領域番号 |
21H02460
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| 研究種目 |
基盤研究(B)
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| 配分区分 | 補助金 |
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| 応募区分 | 一般 |
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| 審査区分 |
小区分43050:ゲノム生物学関連
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| 研究機関 | 京都大学 |
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研究代表者 |
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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| 研究分担者 |
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)
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| 研究期間 (年度) |
2021-04-01 – 2024-03-31
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| 研究課題ステータス |
交付 (2023年度)
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| 配分額 *注記 |
17,420千円 (直接経費: 13,400千円、間接経費: 4,020千円)
2023年度: 5,070千円 (直接経費: 3,900千円、間接経費: 1,170千円)
2022年度: 5,850千円 (直接経費: 4,500千円、間接経費: 1,350千円)
2021年度: 6,500千円 (直接経費: 5,000千円、間接経費: 1,500千円)
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| キーワード | 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 |
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| 研究開始時の研究の概要 |
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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| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
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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| 今後の研究の推進方策 |
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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