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

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 dCas...

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Autores principales: Doi, Goro, Okada, Satoshi, Yasukawa, Takehiro, Sugiyama, Yuki, Bala, Siqin, Miyazaki, Shintaro, Kang, Dongchon, Ito, Takashi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826275/
https://www.ncbi.nlm.nih.gov/pubmed/33398345
http://dx.doi.org/10.1093/nar/gkaa1241
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author Doi, Goro
Okada, Satoshi
Yasukawa, Takehiro
Sugiyama, Yuki
Bala, Siqin
Miyazaki, Shintaro
Kang, Dongchon
Ito, Takashi
author_facet Doi, Goro
Okada, Satoshi
Yasukawa, Takehiro
Sugiyama, Yuki
Bala, Siqin
Miyazaki, Shintaro
Kang, Dongchon
Ito, Takashi
author_sort Doi, Goro
collection PubMed
description 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.
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spelling pubmed-78262752021-01-27 Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability Doi, Goro Okada, Satoshi Yasukawa, Takehiro Sugiyama, Yuki Bala, Siqin Miyazaki, Shintaro Kang, Dongchon Ito, Takashi Nucleic Acids Res Genome Integrity, Repair and Replication 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. Oxford University Press 2021-01-04 /pmc/articles/PMC7826275/ /pubmed/33398345 http://dx.doi.org/10.1093/nar/gkaa1241 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Genome Integrity, Repair and Replication
Doi, Goro
Okada, Satoshi
Yasukawa, Takehiro
Sugiyama, Yuki
Bala, Siqin
Miyazaki, Shintaro
Kang, Dongchon
Ito, Takashi
Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability
title Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability
title_full Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability
title_fullStr Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability
title_full_unstemmed Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability
title_short Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability
title_sort catalytically inactive cas9 impairs dna replication fork progression to induce focal genomic instability
topic Genome Integrity, Repair and Replication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826275/
https://www.ncbi.nlm.nih.gov/pubmed/33398345
http://dx.doi.org/10.1093/nar/gkaa1241
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