研究目的
To develop tools for optical control of the genome to understand and manipulate biological phenomena, including diseases, by merging genome engineering and optogenetics technologies.
研究成果
The developed optical control tools (PA-Cas9, Split-CPTS2.0, PA-Cre) enable precise spatiotemporal manipulation of genome editing and gene expression, expanding applications in life sciences and biomedical research, with potential for understanding gene functions and diseases.
研究不足
The tools rely on blue light irradiation, which may have limited penetration in deep tissues; efficiency and specificity need further optimization; potential off-target effects in genome editing; requires genetic modification of cells or organisms.
1：Experimental Design and Method Selection:
Utilized protein engineering to develop the Magnet photoswitching protein system, applied to split proteins (e.g., split-Cas9, split-dCas9, split-Cre) for optical control.
2：Sample Selection and Data Sources:
Used HEK293T cells and iPS cells for in vitro experiments, and mice for in vivo validation.
3：List of Experimental Equipment and Materials:
Included plasmids, Magnet system proteins, guide RNAs, LED light sources, and EM-CCD camera for imaging.
4：Experimental Procedures and Operational Workflow:
Designed split proteins with Magnet system, transfected cells or injected mice with plasmids, applied blue light irradiation to activate systems, and measured outcomes like gene editing efficiency or luciferase expression.
5：Data Analysis Methods:
Compared efficiencies with control systems (e.g., Cas9 vs. PA-Cas9), used imaging and molecular biology techniques to assess activity.
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