Development of a method to control endogenous proteins by integrating genome editing and chemogenetic techniques

2020 Fiscal Year Final Research Report

• Project/Area Number: 18H02086
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 37010:Bio-related chemistry
• Research Institution: Nagoya Institute of Technology
• Principal Investigator: Tsukiji Shinya 名古屋工業大学, 工学(系)研究科(研究院), 教授 (40359659)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Keywords: 局在性リガンド / ゲノム編集 / 細胞内在性タンパク質 / タンパク質工学 / シグナル制御

Outline of Final Research Achievements

The aim of this work was to develop a new general method for controlling endogenous proteins in living cells. To this end, we took an approach to combine a CRISPR/Cas9-based genome editing technique with our previously established SLIPT (self-localizing ligand-induced protein translocation) method. We first developed a novel engineered dihydrofolate reductase, termed iK6-DHFR, as a protein tag that can be recruited specifically to the plasma membrane (PM) in the presence of a previously reported PM-localizing ligand, mDcTMP. We then established a gene-knock-in protocol to generate cell lines in which a genome-encoded protein of interest is fused in-frame to the iK6-DHFR tag and fluorescent protein. Using cRaf as a target protein, we demonstrated the PM-recruitment of the iK6-DHFR-tagged endogenous cRaf by mDcTMP, which led to the activation of the endogenous Raf/ERK pathway.

Free Research Field

ケミカルバイオロジー

Academic Significance and Societal Importance of the Research Achievements

本研究は、細胞内在性タンパク質の局在と活性を化合物でコントロールすることを可能にする汎用的な手法を開発するものである。このような技術は、細胞や疾患のメカニズムの解明のためのツールとして有用であるばかりでなく、再生医療や細胞治療など、細胞機能の人工制御が基盤となるさまざまなバイオメディカル分野への応用が期待される。