| 研究課題/領域番号 |
23K14545
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| 研究種目 |
若手研究
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| 配分区分 | 基金 |
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| 審査区分 |
小区分49070:免疫学関連
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| 研究機関 | 大阪大学 |
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研究代表者 |
Chen KelvinYigene 大阪大学, 免疫学フロンティア研究センター, 特任助教(常勤) (00898851)
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| 研究期間 (年度) |
2023-04-01 – 2025-03-31
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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 |
4,680千円 (直接経費: 3,600千円、間接経費: 1,080千円)
2024年度: 1,820千円 (直接経費: 1,400千円、間接経費: 420千円)
2023年度: 2,860千円 (直接経費: 2,200千円、間接経費: 660千円)
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| キーワード | Single cell genonmics / Immunology / Thymus development / CRISPR |
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| 研究開始時の研究の概要 |
The applicant proposes the a novel CRISPR screening platform as a powerful tool for understanding phenotypic changes of the transcriptome, epigenome and proteome that can be further extended towards other CRISPR-based applications.
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| 研究実績の概要 |
The purpose of the proposed Research Project was the establishment of a robust platform for screening the effects of hundreds of genetic perturbations at single-cell resolution in vivo. Further, the work aimed to assess the impact of these genetic perturbations in the context of in vivo thymocyte development through DOGMA-seq, a multimodal single-cell genomic assay that simultaneously assess chromatin, RNA transcriptome and select proteins at single cell resolution.
To make the detection of CRISPR perturbations compatible with DOGMA-seq, we adapted CROP-seq with a polyA-based capture to encode for sgRNA identity. We further optimized the CROP-seq lentiviral vector to increase transcript expression by 3-5-fold, enabling much higher sensitivity in gRNA capture compared to the original vector.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
While we initially had some difficulty with efficient capture of the gRNA transcript using the original CROP-seq vector in DOGMA-seq, this issue is now potentially resolved with the development of an optimized vector.
While there are additional parameters that require testing, we are cautiously optimistic that the new vector will help drive the project forward.
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| 今後の研究の推進方策 |
The next phase of this research is to more extensively characterize the novel CROP-seq vectors we generated and then apply them to the in vivo context with DOGMA-seq to assess multi-modal impact of genetic perturbations.
As a general tool for the scientific community, it will be essential to confirm that these new vectors are also compatible with cell types other than lymphocytes as well.
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