Bacteria
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PROVIDER: PRJNA296743 | ENA |
REPOSITORIES: ENA
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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
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:The CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpyCas9) is most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpyCas9), Streptococcus aureus (SauCas9), Lachnospiraceae bacterium (LbaCas12a, previously known as LbCpf1), Acidaminococcus sp. (AspCas12a, previously known as AsCpf1) and Neisseria meningitidis (Nme2Cas9) are orthogonal systems capable of operating simultaneously in zebrafish. We implemented multichannel CRISPR recording using up to three CRISPR systems, and show that LbaCas12a may provide superior information density compared to previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. These results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.
2021-10-25 | GSE186338 | GEO
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:Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.
2023-11-20 | GSE243790 | GEO
Project description:CRISPR loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the cas genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which many have no reported function in CRISPR-mediated immunity. Here, we characterize MM_0565 of Methanosarcina mazei Gö1 associated to the type I-B CRISPR-locus providing evidence for its relevance in regulating this system. We show that MM_0565 is composed of a modified Rossmann-like fold and a winged helix-turn-helix domain and forms a dimer in solution. While direct effects on CRISPR-Cas transcription were not detected by genetic approaches, binding to the leader region of both CRISPR-Cas systems was observed by microscale thermophoresis and electromobility shift assays. Overexpression of MM_0565 however, strongly induced transcription of the cas1-solo gene located in the recently reported casposon, the gene product of which shows high similarity to classical Cas1 proteins. Based on our findings we hypothesize that Cas1-solo is involved in the adaptation of CRISPR-mediated immunity in M. mazei, and that MM_0565 modulates the activity of the CRISPR systems amongst potential other hypnotized actions by activating the transcription of the cas1-solo gene.
2020-09-08 | GSE151372 | GEO
Project description:We report the PAMs of diverse type I-E CRISPR- Cas systems and the type I-C and the type I-F1 CRISPR-Cas systems from Xanthomonas albilineans. Furthermore, we report PAMs of two type I-B CRISPR transposons (CASTs) and the Vibrio cholerae type I-F CAST. For identification of the PAMs, we used a cell-free TXTL-based PAM screen we named PAM-DETECT. By adding a 5N randomized PAM library and plasmids encoding for Cascade genes and gRNAs, recognized PAMs were bound by Cascade and protected from cleavage by a restriction enzyme that has it's recognition site within the target region. By amplifying the non-cleaved target plasmid, we used next-generation sequencing to analyze the enrichment of functional PAMs of the studied CRISPR-Cas systems. We additionally assessed the insertion sites of crRNA-dependent and crRNA-independent transposition of the Rippkaea orientalis type I-B CAST in TXTL and E. coli.
2022-02-24 | GSE179614 | GEO
Project description:NGS data for Systematic Discovery of Novel Type V CRISPR-Cas Systems
| PRJNA496291 | ENA
Project description:New CRISPR-Cas systems from uncultivated microbes
| PRJNA349044 | ENA