Catalytically Inactive Cas9 Impairs DNA Replication Fork Progression to Induce Focal Genomic Instability

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2021 Jan 5

PMID 33398345

Citations 24

Authors

Goro Doi

Satoshi Okada

Takehiro Yasukawa

Yuki Sugiyama

Siqin Bala

Shintaro Miyazaki

Dongchon Kang

Takashi Ito

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

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Repair of replication-dependent double-strand breaks differs between the leading and lagging strands.

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