2023 Fiscal Year Research-status Report
Interrogation of thymocyte development with in vivo CRISPR screens
| Project/Area Number |
23K14545
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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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| Keywords | Single cell genonmics / Immunology / Thymus development / CRISPR |
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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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| Causes of Carryover |
A small portion of the allocated funding could not be used within the first fiscal year due to delays in antibody production and conjugation. However, this does not affect the progress of the research.
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