2021 Fiscal Year Annual Research Report
ゲノム編集細胞の光学スクリーニングによる機能獲得型変異体ライブラリの迅速創出
| Project/Area Number |
21J20659
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| Allocation Type | Single-year Grants |
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| Research Institution | The University of Tokyo |
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Principal Investigator |
Hu Xixun 東京大学, 工学系研究科, 特別研究員(DC1)
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| Project Period (FY) |
2021-04-28 – 2024-03-31
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| Keywords | CRISPR Imaging / genotyping / cell sorting |
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| Outline of Annual Research Achievements |
My research is to promote establishment of genome-edited cell lines for cell therapy, which is by quick cell genotyping and sorting with CRISPR imaging in live cells. My research plan could be divided into three stages, CRISPR imaging, cell sorting and genome editing-based cell therapy. For the first year and the first stage, CRISPR imaging, there’re two goals that I planned to achieve, (1) establishing reliable CRISPR imaging system and (2) optimizing it for further research.
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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
In last year, by comparing with several methods, we established RNP-based CRISPR imaging system in our lab. Genome labeling can be reproducibly achieved in mammalian cells, and CRISPR signals were confirmed by 3D imaging. For optimization step, genome labeling efficiency was increased from ~50% to ~80% by NLS optimization. However, problems about low sensitivity and signal/background ratio are still exist in RNP-based CRISPR imaging, which limits its application and makes it costly. We will keep focusing on improving RNP-based CRISPR imaging system and develop cell sorting technology based on it next year.
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| Strategy for Future Research Activity |
To detect small genomic mutation and genotype based on it, high detection sensitivity is necessary. Thus, we will keep improving signal intensity and signal/background ratio in next year. At the same time, development of CRISPR imaging-based cell selection technology will be scheduled in next year. Large fragments (e.g., chromosome) would be targets in genomic detection by current CRISPR imaging technology first. Detection of small genomic mutations would be scheduled later if we get any progress in system optimization. In summary, we’re planning to perform POC experiments and applications in real world next year.
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