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The autoregulator Aca2 mediates anti-CRISPR repression.

ABSTRACT: CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by the Pectobacterium carotovorum temperate phage ZF40, is an autoregulator that represses the anti-CRISPR-aca2 operon. Aca2 is a helix-turn-helix domain protein that forms a homodimer and interacts with two inverted repeats in the anti-CRISPR promoter. The inverted repeats are similar in sequence but differ in their Aca2 affinity, and we propose that they have evolved to fine-tune, and downregulate, anti-CRISPR production at different stages of the phage life cycle. Specific, high-affinity binding of Aca2 to the first inverted repeat blocks the promoter and induces DNA bending. The second inverted repeat only contributes to repression at high Aca2 concentrations in vivo, and no DNA binding was detectable in vitro. Our investigation reveals the mechanism by which an Aca protein regulates expression of its associated anti-CRISPR.

SUBMITTER: Birkholz N

PROVIDER: S-EPMC6765145 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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The autoregulator Aca2 mediates anti-CRISPR repression.

Birkholz Nils N Fagerlund Robert D RD Smith Leah M LM Jackson Simon A SA Fineran Peter C PC

Nucleic acids research 20191001 18

CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by t ...[more]

PMID: 31428783

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Project description:Tools for tuning endogenous gene expression are key to determining the genetic basis of diverse cellular phenotypes. Although synthetic regulatable promoters are available in Toxoplasma, scalable methods for targeted and combinatorial downregulation of gene expression-like RNA interference-have yet to be developed. To investigate the feasibility of CRISPR-mediated transcriptional regulation, we examined the function of two catalytically inactive Cas9 (dCas9) orthologs, from Streptococcus pyogenes and Streptococcus thermophilus, in Toxoplasma. Following the addition of single-guide RNAs (sgRNAs) targeting the promoter and 5' untranslated region (UTR) of the surface antigen gene SAG1, we profiled changes in protein abundance of targeted genes by flow cytometry for transcriptional reporters and immunoblotting. We found that the dCas9 orthologs generated a range of target gene expression levels, and the degree of repression was durable and stably inherited. Therefore, S. pyogenes and S. thermophilus dCas9 can effectively produce intermediate levels of gene expression in Toxoplasma. The distinct sgRNA scaffold requirements of the two dCas9s permit their orthogonal use for simultaneous examination of two distinct loci through transcriptional modulation, labeling for microscopy-based studies, or other dCas9-based approaches. Taking advantage of newly available genomic transcription start site data, these tools will aid in the development of new loss-of-function screening approaches in Toxoplasma. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular parasite of humans and animals that causes life-threatening disease in immunocompromised patients, fetal abnormalities when contracted during gestation, and recurrent eye lesions in some patients. Despite its health implications, about half of the Toxoplasma genome still lacks functional annotation. A particularly powerful tool for the investigation of an organism's cell biology is the modulation of gene expression, which can produce the subtle phenotypes often required for informing gene function. In Toxoplasma, such tools have limited throughput and versatility. Here, we detail the adaptation of a new set of tools based on CRISPR-Cas9, which allows the targeted downregulation of gene expression in Toxoplasma. With its scalability and adaptability to diverse genomic loci, this approach has the potential to greatly accelerate the functional characterization of the Toxoplasma genome.

Dataset Information

The autoregulator Aca2 mediates anti-CRISPR repression.

Publications

The autoregulator Aca2 mediates anti-CRISPR repression.

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