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

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

Systematic discovery of endogenous ribonucleoprotein complexes - RNA DIF-FRAC Human HEK293T Cells Replicate1

Project description:The associated files are mass spec data from size exclusion chromatographic separations of Human HEK293T with and without RNAse A treatment.

2019-10-18 | PXD015406 | Pride

Systematic discovery of endogenous ribonucleoprotein complexes - RNA DIF-FRAC Human HEK293T Cells Replicate2

Project description:The associated files are mass spec data from size exclusion chromatographic separations of Human HEK293T with and without RNAse A treatment.

2019-10-25 | PXD014820 | Pride

Systematic discovery of endogenous ribonucleoprotein complexes - RNA DIF-FRAC mouse embryonic stem cell

Project description:The associated files are mass spec data from size exclusion chromatographic separations of mouse embryonic stem cells with and without RNAse A treatment.

2019-10-18 | PXD014607 | Pride

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

| PRJNA496616 | ENA