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

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.

SUBMITTER: Doi G

PROVIDER: S-EPMC7826275 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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

Doi Goro G Okada Satoshi S Yasukawa Takehiro T Sugiyama Yuki Y Bala Siqin S Miyazaki Shintaro S Kang Dongchon D Ito Takashi T

Nucleic acids research 20210101 2

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, especia ...[more]

PMID: 33398345

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Project description:There has been an absence of an efficient method of gene knockdown in the important human pathogen Staphylococcus aureus like RNA interference in eukaryotes. The previously developed antisense RNA technology is mainly applied for forward genetic screening but is rather limited in specific gene knockdown because of the lack of rational antisense RNA design strategies. Here we report an efficient and specific system for gene knockdown in S. aureus based on the type II clustered regularly interspaced short palindromic repeat (CRISPR) system from Streptococcus pyogenes We can achieve gene silencing with the coexpression of dCas9, an RNA-guided DNA binding protein, and a small guide RNA complementary to the target gene. With this system, we have successfully silenced diverse sets of genes varying in size and expression level in different S. aureus strains. This system exhibited high-efficiency knockdown of both essential and nonessential genes, and its effect is inducible and reversible. In addition, the system can repress the expression of multiple genes simultaneously and silence an entire operon or part of it. This RNA-guided DNA targeting system thus provides a simple, rapid, and affordable method for selective gene knockdown in S. aureusIMPORTANCEStaphylococcus aureus is an important human and animal pathogen that can cause a diversity of infectious diseases. Molecular genetic study of S. aureus has provided an avenue for the understanding of its virulence, pathogenesis, and drug resistance, leading to the discovery of new therapies for the treatment of staphylococcal infections. However, methodologies developed for genetic manipulation of S. aureus usually involve either low efficiency or laborious procedures. Here we report an RNA-guided system for gene knockdown in S. aureus and show its high efficiency and simplicity for selective gene silencing in different strains of S. aureus This simple, rapid, and affordable system may serve as a promising tool for functional gene study in S. aureus, especially for the study of essential genes, thus facilitating the understanding of this pathogen and its interaction with its hosts.

Dataset Information

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

Publications

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

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