| Project/Area Number |
23K14545
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 49070:Immunology-related
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| Research Institution | Osaka University |
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Principal Investigator |
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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| Project Period (FY) |
2023-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2024: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2023: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
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| Keywords | Single cell genonmics / Immunology / Thymus development / CRISPR |
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| Outline of Research at the Start |
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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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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