Catalytically inactive Cas9-induced genome destabilization: mechanistic study and applications to manipulation of structural variations

2020 Fiscal Year Final Research Report

• Project/Area Number: 19K22397
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: Kyushu University
• Principal Investigator: Ito Takashi 九州大学, 医学研究院, 教授 (90201326)
• Project Period (FY): 2019-06-28 – 2021-03-31
• Keywords: dCas9 / ゲノム不安定性 / 複製フォーク停止 / 構造多型 / 組換え修復

Outline of Final Research Achievements

Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. Here we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ~16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.

Free Research Field

ゲノム科学

Academic Significance and Societal Importance of the Research Achievements

dCas9が複製フォークの進行を阻害して、ゲノムを不安定化させることを見い出した。これによって、ゲノム中の任意の部位で複製フォークを停止させる途が示された。dCas9によるゲノム不安定性の誘導は、ゲノム安定性研究における新しい研究手法を提供するとともに、構造多型を誘導する新しいゲノム操作技術の礎となることが期待される。また、様々な応用が試みられているdCas9の潜在的危険性を示したことにより、dCas9の様々な社会的応用の安全性向上にも貢献することが期待される。