Elucidation of off-target effects mechanisms of action using anti-CRISPR molecules for genome editing safety

2021 Fiscal Year Final Research Report

• Project/Area Number: 19H02827
• 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: Hiroshima University
• Principal Investigator: Nomura Wataru 広島大学, 医系科学研究科(薬), 教授 (80463909)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: ゲノム編集 / CRISPR-Cas / アンチクリスパー / オフターゲット作用 / タンパク質間相互作用

Outline of Final Research Achievements

Regarding the cell cycle-dependent genome editing method that enhances the accuracy and safety of genome editing technology, we succeeded in observing the interaction timing of anti-CRISPR and Cas9 in cells using transcriptional activation by dCas9-VPR. The results also showed that accurate genome editing can be performed on multiple target genes, and that a wide range of applications can be expected. We also examined the use of Geminin, which shows different expression timing from Cdt1, and the usefulness of Cdt1 was once again confirmed. But we also obtained suggestions on how to use Geminin in future applications. We also examined the in vivo application of cell cycle-dependent genome editing by incorporating SaCas9, which can be carried by adeno-associated viruses.

Free Research Field

生物化学

Academic Significance and Societal Importance of the Research Achievements

オフターゲット作用の低減に関する研究は国外のグループによる開発競争が激化しており、特に安全面が重要となる医療分野、育種・品種改良分野での応用においては非常に重要な要素技術になる。今回の成果で、anti-CRISPRを利用したオフターゲット作用の抑制について、細胞内でのCas9との相互作用メカニズムを明らかできたことで、さらに正確性の高いゲノム編集法へと改良できる可能性が示された。anti-CRISPRの医療や産業への応用は比較的進んでいないため、正確性の高いゲノム編集法のin vivoなどへの応用だけでなく、新しい活用方法にもつながる成果が得られたと考えられる。