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Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

ABSTRACT: Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

PROVIDER: PRJEB12161 | ENA |

REPOSITORIES: ENA

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Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.

2016-05-01 | E-MTAB-4143 | biostudies-arrayexpress

Systematic discovery of endogenous human ribonucleoprotein complexes

Project description:An exploration of human protein complexes in the presence and absence of RNA reveals endogenous ribonucleoprotein complexes

2019-09-19 | GSE137651 | GEO

Systematic discovery of endogenous human ribonucleoprotein complexes

Project description:Systematic discovery of endogenous human ribonucleoprotein complexes

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C. elegans DLC-1 is a component of diverse ribonucleoprotein complexes

Project description:Ribonucleoprotein complexes, which contain mRNAs and their regulator proteins, carry out post-transcriptional control of gene expression. The function of many RNA-binding proteins depends on their association with cofactors. Here we use a genomic approach to identify transcripts associated with DLC-1, a protein previously identified as a cofactor of two unrelated RNA-binding proteins that act in the C. elegans germline. Among the 2732 potential DLC-1 targets, most are germline mRNAs. Removal of DLC-1 affects expression of its targets, meg-1 and meg-3. We propose that DLC-1 acts as a cofactor for multiple ribonucleoprotein complexes, thus making diverse contributions to RNA regulation in the germline.

2018-10-01 | GSE115281 | GEO

C. elegans DLC-1 is a component of diverse ribonucleoprotein complexes

Project description:C. elegans DLC-1 is a component of diverse ribonucleoprotein complexes

| PRJNA474476 | ENA

The scaffolding role of RNA in ribonucleoprotein complexes: TRA2A recruitment in FXTAS disease

Project description:The scaffolding role of RNA in ribonucleoprotein complexes: TRA2A recruitment in FXTAS disease

| PRJNA422212 | ENA

The scaffolding role of RNA in ribonucleoprotein complexes: TRA2A recruitment in FXTAS disease

Project description:The scaffolding role of RNA in ribonucleoprotein complexes: TRA2A recruitment in FXTAS disease

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Pre-assembled Cas9 ribonucleoprotein-mediated gene deletion identifies the carbon catabolite repressor and its target genes in Coprinopsis cinerea

Project description:Cre1 is an important transcription factor that regulates carbon catabolite repression (CCR) and is widely conserved across fungi. This gene has been extensively studied in several Ascomycota species, whereas its role in gene expression regulation in the Basidiomycota remains poorly understood. Here, we identified and investigated the role of cre1 in Coprinopsis cinerea, a basidiomycete model mushroom that can efficiently degrade lignocellulosic plant wastes. We used a rapid and efficient gene deletion approach based on PCR-amplified split-marker DNA cassettes together with in-vitro assembled Cas9-guide RNA ribonucleoproteins (Cas9-RNPs) to generate C. cinerea cre1 gene deletion strains. Gene expression profiling of two independent C. cinerea cre1 mutants showed significant deregulation of carbohydrate metabolism, plant cell wall degrading enzymes (PCWDEs), plasma membrane transporter-related and several transcription factor encoding genes, among others. Our results support the notion that, similarly to reports in the ascomycetes, Cre1 of C. cinerea orchestrates CCR through a combined regulation of diverse genes, including PCWDEs, transcription factors that positively regulate PCWDEs and membrane transporters which could import simple sugars that can induce the expression of PWCDEs. Somewhat paradoxically, though in accordance with other Agaricomycetes, genes related to lignin degradation were mostly downregulated in cre1 mutants, indicating they fall under different regulation than other PCWDEs. The gene deletion approach and the data presented in this paper expand our knowledge of CCR in the Basidiomycota and provide functional hypotheses on genes related to plant biomass degradation.

2022-11-01 | GSE205749 | GEO

Development of microtiter plate scale CRISPR/Cas9 transformation method for <i>Aspergillus niger</i> based on in vitro assembled ribonucleoprotein complexes.

Project description: Not available

| S-EPMC6419801 | biostudies-literature

Trf4p in vivo crosslinking and ribonucleoprotein-immunopurification-chip analysis (X-RIP-Chip)

Project description:In vivo cross-linking and ribonucleoprotein-immunopurification experiments followed by microarray analysis of bound RNAs (X-RIP-chip). Cells expressing recombinant tandem-affinity purification (TAP)-tagged Trf4 protein were cross-linked with formaldehyde, and Trf4-containing ribonucleoprotein complexes were recovered by affinity selection on IgG-coupled beads (see linked protocol). As a control for non-specifically enriched RNAs, the same experiment was done with untagged WT cells and with cells expressing Fpr1-TAP, a peptidyl-prolyl-cis-trans-isomerase not expected to bind RNA. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Antigenic peptide used in IP: Protein A derivative Computed

2010-06-24 | E-GEOD-16105 | biostudies-arrayexpress

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