Efforts toward precise and efficient homologous recombination in genome editing.

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K07093
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Laboratory animal science
• Research Institution: Kumamoto University
• Principal Investigator: Araki Kimi 熊本大学, 生命資源研究・支援センター, 教授 (90211705)
• Co-Investigator(Kenkyū-buntansha): 荒木 正健 熊本大学, 生命資源研究・支援センター, 准教授 (80271609)
• Research Collaborator: TAKEDA Naoki
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: ゲノム編集 / 相同組換え / ES細胞

Outline of Final Research Achievements

Genome editing using CRISPR / Cas9 is an essential tool for producing genetically modified mice. However, when double strand breaks (DSBs) are introduced on the genome DNA to induce homologous recombination (HR)in mouse ES cells, although the rate of successful HR is dramatically increased, alleles in which HR have not occurred frequently show gross rearrangement. We developed a system that can distinguish between accurate gene targeting by HR and gene targeting with rearrangement in ES cells. We examined the use of Cas9 D10A nickase, which rarely induce rearrangement, and found that multiple nicks on the same strand is effective, although the efficient of HR was lower than that of DSB.

Free Research Field

発生工学

Academic Significance and Societal Importance of the Research Achievements

ゲノム編集は、DNAを目的の場所で2本鎖切断(DSB)することで遺伝子の改変を効率よく行う技術で、遺伝子改変マウス作製効率は飛躍的に上昇した。しかし、DSBを加えると、２つある対立アレルのうち、目的通りの遺伝子改変が起こらなかった対立アレルでは、大きなリアレンジが高い頻度で生じてしまう。そのような細胞やマウスは解析に適さないため、リアレンジを抑える技術の開発は解析効率を上げるために重要である。１本鎖切断を起こすCas9 D10A nickaseを効果的に利用できることがわかったので、マウスES細胞を用いた遺伝子改変を安全かつ迅速に進めることができるようになった。