| 研究課題/領域番号 |
21H02460
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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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| キーワード | ゲノム編集 / ゲノム解析 / DNAリピート / ヒトiPS細胞 |
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| 研究実績の概要 |
Using repeat finder and MHcut software, we generated a dataset of repetitive DNA sequences and deletion variants overlapping candidate regulatory elements. We used genomic conservation to select human- and primate-specific loci for gene editing. Initial gene editing experiments aimed to generate variants in select repetitive loci already associated with evolution or neurological disease. All CRISPR gene editing was performed in normal isogenic human iPSC lines. We established protocols for mRNA transfection of neurogenin-2 (NGN2) expression for efficient and homogenous differentiation of excitatory neurons. We began to design a lentivirus-based library for massively parallel reporter assay (MPRA) to evaluate the activity of enhancers and their polymorphic deletion variants.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Preliminary preparations, genome data investigation and assay library design proceeded as scheduled during FY2021. Construction of the assay library, application of the assay library, gene editing and compilation of research results were scheduled to be carried out by the end FY2021. We faced difficulty in constructing libraries essential for continuing this aspect of the project due to restrictions for in-person visits to collaborating institutions. To advance the project, we made efforts to develop editing and genotyping strategies for VNTRs identified from the literature to be associated with disease.
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| 今後の研究の推進方策 |
Using our computational analysis of repeat variants overlapping candidate regulatory elements, we will construct a lentivirus-based reporter assay library for evaluating the activity of enhancers and their polymorphic deletion variants. We will use lentivirus-based massively parallel reporter assay (MPRA) technology for high-throughput screening of enhancer variants in iPSCs and neurons. Reporter-positive cells will be binned and recovered. Transcribed barcodes detected in single-cell RNA-seq data will identify VNTRs encoding active cCREs. MPRA will allow us to classify and prioritize variants for characterization using primate ESCs or iPSC-derived in vitro models. Additionally, genomic conservation will be used to select human- and primate-specific loci for gene editing.
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