A high-content arrayed CRISPR screen reveals genetic requirements for dynein-based trafficking
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE218249 | GEO | 2024/02/29
REPOSITORIES: GEO
Similar Datasets
Project description:A high-content arrayed CRISPR screen reveals genetic requirements for dynein-based trafficking
| PRJNA902801 | 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:RNA-sequencing data from HEK293T and HAP1 cell models: Hek293T (SF3B1-K700E, SF3B1-WT, SUGP1-KD and siContol), HAP1 CRISPR-isogenic cell lines WT and SUGP1-P636L
2020-10-10 | GSE159304 | 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 still restrained by suboptimal efficiencies. Here, we show that U1-driven CRISPR RNAs (crRNAs) can dramatically increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effectors. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined in the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of different 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-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: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
Project description:In bacteria and archaea, CRISPR loci confer adaptive, sequence-based immunity against viruses and plasmids. CRISPR interference is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from CRISPR spacers and repeats. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here we examine crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp., including the human pathogen N. meningitidis. Our studies reveal a unique crRNA maturation pathway in which crRNA transcription is driven by promoters that are embedded within each repeat, yielding crRNA 5’ ends are not formed by processing. Although crRNA 3’ end formation occurs through RNase III cleavage of a pre-crRNA/tracrRNA duplex, as in other Type II CRISPR systems, this processing event is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/cas system characterized to date. Endogenous CRISPR spacers frequently target genomic sequences of other Neisseria strains and so limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in N. meningitidis. dRNA-seq approach for RNA samples from cultures of N. lactamica 020-06, harvested at mid-log. Two cDNA libraries from total RNA were prepared to distinguish between transcripts with either primary orprocessed 5’ ends: one library is generated from untreated RNA, whereas the other is treated with terminator exonuclease (TEX),
2013-04-23 | E-GEOD-44582 | biostudies-arrayexpress
Project description:The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.
| S-EPMC10916854 | biostudies-literature
Project description:We measured global RNA levels using RNA-seq in cas3+ E. coli cells with intact CRISPR arrays or cells with the CRISPR-I array deleted to determine if off-target events driven by endogenous spacers affect RNA levels.
2018-02-10 | E-MTAB-6446 | biostudies-arrayexpress
Project description:We measured global RNA levels using RNA-seq in cas3 E. coli cells with intact CRISPR arrays or cells with the CRISPR-I array deleted to determine if off-target events driven by endogenous spacers affect local gene expression.
2017-08-24 | E-MTAB-5971 | biostudies-arrayexpress