Development of intercellular device to record the extracellular stimuli with self-targeting CRISPR-Cas9

2018 Fiscal Year Final Research Report

• Project/Area Number: 17K15045
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Medical genome science
• Research Institution: Hiroshima University
• Principal Investigator: Nakade Shota 広島大学, 理学研究科, 研究員 (70795509)
• Project Period (FY): 2017-04-01 – 2019-03-31
• Keywords: ゲノム編集 / CRISPR-Cas9 / がん細胞 / スクリーニング / ゲノム / 癌

Outline of Final Research Achievements

In this research, we established the efficient method of genome editing serving as a platform for knock-out screening with CRISPR-Cas9 in breast cancer cells, which is called Local Accumulation of DSB repair molecules (LoAD). It enabled efficient gene modification by accumulating double-strand break (DSB) repair molecules into the genome editing region. We achieved the improvement of gene knock-in, the practical application of simultaneous triple knock-in, and induction of short deletion through LoAD method. Also, we provided the Cas9-expressing stable cell lines of breast cancer cells using the lentiviral vector for CRISPR Screening.

Free Research Field

ゲノム編集

Academic Significance and Societal Importance of the Research Achievements

LoAD法の開発によって、ゲノム編集によるノックイン効率を大きく上昇させることに成功した。これは遺伝子改変細胞株の樹立効率を引き上げ、ゲノム編集を用いた遺伝学的解析に必要な期間を大幅に短縮する。また、CRISPR-Cas9の変異導入効率や遺伝子導入効率が低い細胞種でも改変株作製が高効率に可能になった。さらに同法は、局在化させるDSB 修復遺伝子をカスタマイズすることで、オーダーメイドな遺伝子改変を誘導する手法の発展に繋がる。