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Tuning dCas9's ability to block transcription enables robust, noiseless knockdown of bacterial genes.

ABSTRACT: Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase "kicks out" dCas9 from the target and completes transcription. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. Alternatively, tuning repression by changing dCas9 concentration is noisy and promoter-strength dependent. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of amount variations of cell-wall synthesizing enzymes on cell morphology. Finally, we multiplex the system to obtain any combination of fractional repression of two genes.

SUBMITTER: Vigouroux A

PROVIDER: S-EPMC5842579 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Tuning dCas9's ability to block transcription enables robust, noiseless knockdown of bacterial genes.

Vigouroux Antoine A Oldewurtel Enno E Cui Lun L Bikard David D van Teeffelen Sven S

Molecular systems biology 20180308 3

Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase "kicks out" dCas9 from the target and completes transcription. We use ...[more]

PMID: 29519933

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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

Tuning dCas9's ability to block transcription enables robust, noiseless knockdown of bacterial genes.

Publications

Tuning dCas9's ability to block transcription enables robust, noiseless knockdown of bacterial genes.

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