Similar Datasets
Project description:In this work, we analyzed recognized PAM sequences from four CRISPR-Cas systems: E. coli I-E, B. halodurans I-C, S. thermophilus CR1 II-A, and F. novicida V. Cells containing functional PAMs were sorted using FACS and subsequently sequenced. Ranking of recognized PAMs from a positive screen
2016-02-17 | E-GEOD-75718 | biostudies-arrayexpress
Project description:In this work, we analyzed recognized PAM sequences from four CRISPR-Cas systems: E. coli I-E, B. halodurans I-C, S. thermophilus CR1 II-A, and F. novicida V. Cells containing functional PAMs were sorted using FACS and subsequently sequenced.
2016-02-17 | GSE75718 | GEO
Project description:In this work the favorable PAM sequences for a type IV-A CRISPR-Cas system were determined
2022-08-19 | GSE211446 | 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:Our results show that compared to wild type, a deletion mutant of the cas3 gene, an essential nuclease part of the class 1 type I CRISPR-Cas system, increases the virulence of P gingivalis.
2020-09-16 | GSE154569 | GEO
Project description:The type V-I CRISPR-Cas system is becoming increasingly attractive for its potential utility in gene editing. However, natural nucleases often exhibit low efficiency, limiting their application. Here, we utilized structure-guided rational design and combinatorial protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. Accordingly, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene-editing activity in mammalian cells and plants. Cas-SF01 displays comparable or superior editing performance compared to SpCas9 or recently engineered Cas12 nucleases. Further analysis of PAM recognition showed that Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. Additionally, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we demonstrated that Cas-SF01 has robust gene-editing activity in both the monocot plant rice and dicot plant pepper. Our results suggest that Cas-SF01 can serve as a robust gene-editing platform with high efficiency and specificity for future genome editing applications across different organisms.
2023-11-05 | GSE236755 | GEO
Project description:Plasmids were constructed harboring protospacers that are perfect targets for spacers #4 and #21 of the array, but contain NNN (all possible PAM combinations) immediately upstream of the protospacer. The plasmids were introduced into V. cholerae with or without a functional CRISPR-cas system and cells were plated on selective media. Cells were collected and the protospacer plasmids were sequenced in a high throughput manner. PAMs were counted using a custom python script
2024-05-06 | E-MTAB-14044 | biostudies-arrayexpress
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:In this work we analyzed crRNA processing by SuCasΩ Type V CRISPR-Cas nuclease in E. coli
2023-01-11 | GSE178531 | GEO
Project description:New, orthogonal transcription factors in eukaryotic cells have been realized by engineering nuclease-deficient CRISPR-associated proteins and/or their guide RNAs. In this work, we present a new kind of orthogonal activators, in Saccharomyces cerevisiae, made by turning type V CRISPR RNA into a scaffold RNA (ScRNA) able to recruit a variable number of VP64 activation domains. The activator arises from the complex between the synthetic ScRNA and DNase-deficient type V Cas proteins: dCas12e and denAsCas12a. The transcription activation achieved via the newly engineered dCas:ScRNA system is up to 4.7-fold higher than that obtained with the direct fusion of VP64 to Cas proteins. The new transcription factors have been proven to be functional in circuits such as Boolean gates, converters, multiplex-gene and metabolic-pathway activation. Our results extend the CRISPR-Cas-based technology with a new effective tool that only demands RNA engineering and improves the current design of transcription factors based on type V Cas proteins.
2023-12-05 | GSE249355 | GEO