Screening for factors that enhance HDR by CRISPR-Cas9

2021 Fiscal Year Final Research Report

• Project/Area Number: 19K06631
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43060:System genome science-related
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: KATO-INUI Tomoko 公益財団法人東京都医学総合研究所, 疾患制御研究分野, 研究員 (10638802)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: 相同組換え修復 / HDR / CRISPR-Cas9 / スクリーニング

Outline of Final Research Achievements

CRISPR-Cas9 is a versatile technology that can be applied to medical treatment. Between the repair pathway that occur after DNA double-stranded breaks, repair by homologous recombination with template DNA (HDR) contributes to precise editing, but at the same time NHEJ, which involves in base deletions or insertions, also occurs at a high frequency. I performed the cell-based screening for HDR-enhancing factors using the Traffic Light Reporter system, which can simultaneously detect cells with HDR and NHEJ, and identified several genes that are highly expressed in HDR-derived cells compared to NHEJ-derived cells. Further gene ontology analysis of these genes suggested that they are related to DNA repair and the cell cycle.

Free Research Field

ゲノム編集

Academic Significance and Societal Importance of the Research Achievements

本研究は正確なゲノム編集技術の確立を目指すと同時に、CRISPR-Cas9の分子機構の解明を更に加速させる可能性を秘めている。特に、HDR効率の高い哺乳類細胞からcDNAライブラリを用いたゲノムワイドな研究と、HDRとNHEJの同時検出を可能にするレポーター遺伝子を用いたスクリーニングの組み合わせによる研究は画期的な研究手法である。既存の概念を打破した研究への展開には、CRISPRスクリーニングは必須のツールであり、本研究により正確なゲノム編集が達成できれば、国内外問わず、ゲノム編集の実用化に向けての礎となる重要な成果となる。