Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability

Goro Doi, Satoshi Okada, Takehiro Yasukawa, Yuki Sugiyama, Siqin Bala, Shintaro Miyazaki, Dongchon Kang, Takashi Ito

研究成果: Contribution to journalArticle査読

1 被引用数 (Scopus)

抄録

Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. 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.

本文言語英語
ページ(範囲)954-968
ページ数15
ジャーナルNucleic acids research
49
2
DOI
出版ステータス出版済み - 1 25 2021
外部発表はい

All Science Journal Classification (ASJC) codes

  • 遺伝学

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