CRISPR/dCas-based modulation of bacterial IS-transposition
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ABSTRACT:
PROVIDER: PRJNA435096 | ENA |
REPOSITORIES: ENA
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Project description:In this work, a plasmid-based system is applied to inhibit the transposition of bacterial insertion sequences (IS). Using multiple guide RNAs, inactivated Cas9 was directed to simultaneously bind to the left end of IS1, IS5, IS3 and IS150 in Escherichia coli, in vivo. As a result, the transcription of IS1, and IS5 was successfully silenced, in certain cases by two orders of magnitude. The transposition rate of all four targeted elements nevertheless dropped to negligible levels, as verified at the cycA and bgl chromosomal loci. A GFP-expressing plasmid, known to be predominantly inactivated by insertion mutations also displayed a significant increase in stability. The transposition-silencing effect was easily transferable between various E. coli strains by plasmid transformation. Our portable system, or other plasmids constructed likewise can serve as useful tools to eliminate insertion mutagenesis or selectively study distinct transposable elements in numerous prokaryotic species.
2019-02-21 | GSE110946 | GEO
Project description:Bacterial transposons are pervasive mobile genetic elements that exploit distinct DNA binding proteins for their horizontal spread. For example, E. coli Tn7 homes to a specific attachment site using a TniQ family protein, whereas diverse Tn7-like transposons employ Type I or Type V CRISPR-Cas systems to insert downstream of target sites specified by a guide RNA. Despite this targeting pathway diversity, transposition invariably requires TnsB, a DDE superfamily transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between the transposase and targeting proteins. How TnsC mediates this communication and thereby regulates transposition fidelity has remained elusive. Here we apply chromatin immunoprecipitation sequencing (ChIP-seq) to monitor in vivo formation of the Vibrio cholerae RNA-guided transpososome, allowing us to unambiguously resolve distinct protein recruitment events prior to integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous, leading to binding at hundreds of genomic off-target sites, but only a subset of those sites are licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint that controls transposition fidelity. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we analysed V. cholerae TnsC by cryo-EM and found that TnsC forms ATP-dependent heptameric rings, which are likely to play a critical architectural role in positioning DNA substrates for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive binding of distinct factors to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.
2021-10-31 | GSE183114 | GEO
Project description:Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.
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Project description:To determine whether CRISPR interference can be used to modulation glycosylation in Burkholderia cenocepacia
2023-03-20 | PXD036564 | Pride
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2023-03-20 | PXD036619 | Pride
Project description:CRISPR-based molecular recording of mouse embryogenesis
| PRJNA482431 | ENA
Project description:The goal of these experiments was to define the targets of Ty3 transposition in Saccharomyces cerevisiae. Ty3 is a retroviruslike element that is found at the transcription initiation site of chromosomal tRNA genes. A Ty3 that can be induced by growth in galactose-containing medium and which was marked by an insertion of HIS3 downstream of the second open reading frame of the element (POL3) was induced to undergo transposition by plating cells onto galactose containing medium and replica-plating onto medium selective for cells that had undergone transposition. These cells were collected, DNA was extracted, and inverse PCR was performed using primers inside the Ty3 element in order to generate a library of insertion sites flanked by Illumina sequence-compatible primers.
2011-09-02 | E-GEOD-31820 | biostudies-arrayexpress
Project description:We used ChIP-seq to map binding of the CRISPR surveillance complex, Cascade, in a Salmonella enterica serovar Typhimurium strain lacking the gene encoding the endonuclease Cas3. We performed ChIP-seq in strains with wild-type and mutant sequences upstream of the two CRISPR arrays, and in strains with wild-type and mutant nusE genes to determine the impact of Nus factor antitermination on CRISPR array function.
2018-10-13 | E-MTAB-7242 | biostudies-arrayexpress
Project description:The development of CRISPR-Cas systems for targeting DNA and RNA in diverse organisms has transformed biotechnology and biological research. Moreover, the CRISPR revolution has highlighted bacterial adaptive immune systems as a rich and largely unexplored frontier for discovery of new genome engineering technologies. In particular, the class 2 CRISPR-Cas systems, which use single RNA-guided DNA-targeting nucleases such as Cas9, have been widely applied for targeting DNA sequences in eukaryotic genomes. Here, we report DNA-targeting and transcriptional control with class I CRISPR-Cas systems. Specifically, we repurpose the effector complex from type I variants of class 1 CRISPR-Cas systems, the most prevalent CRISPR loci in nature, that target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation, and nuclear localization in human cells and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering transactivation domains to Cascade, we modulate the expression of targeted chromosomal genes in both human cells and plants. This study expands the toolbox for engineering eukaryotic genomes and establishes Cascade as a novel CRISPR-based technology for targeted eukaryotic gene regulation.
2019-06-26 | GSE114858 | GEO
Project description:The development of CRISPR-Cas systems for targeting DNA and RNA in diverse organisms has transformed biotechnology and biological research. Moreover, the CRISPR revolution has highlighted bacterial adaptive immune systems as a rich and largely unexplored frontier for discovery of new genome engineering technologies. In particular, the class 2 CRISPR-Cas systems, which use single RNA-guided DNA-targeting nucleases such as Cas9, have been widely applied for targeting DNA sequences in eukaryotic genomes. Here, we report DNA-targeting and transcriptional control with class I CRISPR-Cas systems. Specifically, we repurpose the effector complex from type I variants of class 1 CRISPR-Cas systems, the most prevalent CRISPR loci in nature, that target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation, and nuclear localization in human cells and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering transactivation domains to Cascade, we modulate the expression of targeted chromosomal genes in both human cells and plants. This study expands the toolbox for engineering eukaryotic genomes and establishes Cascade as a novel CRISPR-based technology for targeted eukaryotic gene regulation.
2019-06-26 | GSE114857 | GEO