A dynamic subpopulation of CRISPR-Cas overexpressers allows Streptococcus pyogenes to rapidly respond to phage
Ontology highlight
ABSTRACT:
ORGANISM(S): Streptococcus pyogenes
PROVIDER: GSE263698 | GEO | 2024/05/14
REPOSITORIES: GEO
Similar Datasets
Project description:Cas9 is a transcriptional autoregulator in many strains of S. pyogenes. A natural single guide RNA (tracr-L) directs Cas9 to bind to and repress its promoter. In this study, we aimed to determine whether tracr-L, crRNAs, or other noncanonical guide RNAs direct Cas9 to bind to other regions of the genome and potentially regulate the expression of additional genes.
2024-05-14 | GSE263699 | GEO
Project description:A dynamic subpopulation of CRISPR-Cas overexpressers allows Streptococcus pyogenes to rapidly respond to phage
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Project description:A dynamic subpopulation of CRISPR-Cas overexpressers allows Streptococcus pyogenes to rapidly respond to phage [RNA-seq]
| PRJNA1098752 | ENA
Project description:CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism. 4 Samples: Protein-associated small RNAs
2015-07-10 | E-GEOD-65781 | biostudies-arrayexpress
Project description:CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism.
2015-07-10 | GSE65781 | GEO
Project description:Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are restrained by suboptimal efficiencies. Here, we show that U1 promoter-driven CRISPR RNAs (crRNAs) increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effector. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined to the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.
2024-09-15 | GSE275245 | GEO
Project description:CRISPR-Cas9 expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells. To investigate whether Cas9 can interact with endogenous human RNA transcripts independent of its guide, we perform eCLIP (enhanced CLIP) of Cas9 in human cells and find that Cas9 reproducibly interacts with hundreds of endogenous human RNA transcripts. This association can be partially explained by a model built on gRNA secondary structure and sequence. Critically, transcriptome-wide Cas9 binding sites do not appear to correlate with published genome-wide Cas9 DNA binding or cut-site loci under gRNA co-expression. However, even under gRNA co-expression low-affinity Cas9-human RNA interactions (which we term CRISPR crosstalk) do correlate with published elevated transcriptome-wide RNA editing. Our findings do not support the hypothesis that human RNAs can broadly guide Cas9 to bind and cleave human genomic DNA, but they illustrate a clear cellular RNA impact likely inherent to CRISPR-Cas systems.
2022-01-24 | GSE167466 | GEO
Project description:Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR⁻Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR⁻Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR⁻Cas systems, specifically the three major types (I, II, and III), can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR⁻Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms.
| S-EPMC6024849 | biostudies-literature
Project description:Bacteriophages exert strong evolutionary pressure on their microbial hosts. In their lytic lifecycle, complete bacterial subpopulations are utilized as hosts for bacteriophage replication. However, during their lysogenic lifecycle, bacteriophages can integrate into the host chromosome and alter the host's genomic make-up, possibly resulting in evolutionary important adjustments. Not surprisingly, bacteria have evolved sophisticated immune systems to protect against phage infection. Streptococcus pyogenes isolates are frequently lysogenic and their prophages have been shown to be major contributors to the virulence of this pathogen. Most S. pyogenes phage research has focused on genomic prophages in relation to virulence, but little is known about the defensive arsenal of S. pyogenes against lytic phage infection. Here, we characterized Phage A1, an S. pyogenes bacteriophage, and investigated several mechanisms that S. pyogenes utilizes to protect itself against phage predation. We show that Phage A1 belongs to the Siphoviridae family and contains a circular double-stranded DNA genome that follows a modular organization described for other streptococcal phages. After infection, the Phage A1 genome can be detected in isolated S. pyogenes survivor strains, which enables the survival of the bacterial host and Phage A1 resistance. Furthermore, we demonstrate that the type II-A CRISPR-Cas system of S. pyogenes acquires new spacers upon phage infection, which are increasingly detectable in the absence of a capsule. Lastly, we show that S. pyogenes produces membrane vesicles that bind to phages, thereby limiting the pool of phages available for infection. Altogether, this work provides novel insight into survival strategies employed by S. pyogenes to combat phage predation.
| S-EPMC8066415 | biostudies-literature
Project description:CRISPR/Cas loci commonly encode both proteins and small guide RNAs (crRNAs) to assemble ribonucleoproteins particles (RNPs) that confer a sequence-based, adaptive immunity against viruses and plasmids in prokaryotes. However, it has not been established whether this conserved synteny of RNA and protein genes is needed for the efficient RNP production in vivo. We show that the pathogenic bacterium Salmonella Typhimurium harbours two physically unlinked loci, CRISPR01 and CRISPR02, both of which produce mature crRNAs, although CRISPR02 lacks the protein genes for the type I-specific Cascade complex. Utilizing these coexisting complete and orphan CRISPR loci, we provide the first in vivo evidence for a crRNA-dependent assembly of Cascade. Strikingly, the full RNP assembles identically on crRNAs from either CRISPR cassette, i.e. regardless of their expression in cis or trans. Deep sequencing of Cas-bound transcripts identifies crRNAs derived from both loci as the sole bona fide targets of Cascade, although individual Cas proteins may interact with other cellular RNAs outside of the Cascade context. Altogether, our data demonstrate that CRISPR RNAs, independently of their origin, serve as the organizing centre of Cascade in vivo and are the main reason-to-be for its protein components in Salmonella.
2017-04-24 | GSE58602 | GEO