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IPSC RNA-seq upon CRISPRn or CRISPRi manipulation

ABSTRACT: Assess the on- and off-target effects of dox-inducible CRISPR/Cas9 and CRISPRi constructs in a human iPS cell line.

ORGANISM(S): Homo sapiens

PROVIDER: GSE76980 | GEO | 2016/01/19

REPOSITORIES: GEO

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iPSC RNA-seq upon CRISPRn or CRISPRi manipulation

Project description:Assess the on- and off-target effects of dox-inducible CRISPR/Cas9 and CRISPRi constructs in a human iPS cell line. Transcript quantification of 3 cell lines, each plus or minus doxycycline and with or without specific single guide RNAs (sgRNAs), with 2 biological replicates each.

2016-01-19 | E-GEOD-76980 | biostudies-arrayexpress

Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

Project description:Comparisons of molecular phenotypes across primates provide unique information to understand human biology and evolution and single-cell RNA-seq CRISPR interference screens are a powerful approach to analyze them. Here, we generate and validate three human, three gorilla and two cynomolgus iPS cell lines that carry a dox-inducible KRAB-dCas9 construct in the AAVS1 locus. We show that despite variable expression levels of KRAB-dCas9 among lines, comparable downregulation of target genes and comparable phenotypic effects are observed in a single-cell RNA-seq CRISPR interference screen. Hence, we provide valuable resources for performing and further extending CRISPRi screens in human and non-human primates.

2024-03-26 | GSE241293 | GEO

Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

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Functional Significance of U2AF1 S34F Mutation in Lung Adenocarcinomas

Project description:We conducted RNA-Seq and iCLIP for dox-inducible constructs of FLAG-HA tagged U2AF1 in the wild-type and mutant forms.

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RNA-seq of CSMD1 enhancer CRISPRi in LUHMES cells and differentiated neurons

Project description:we performed lentiviral CRISPR interference (CRISPRi) by recruiting dCas9 fused with the KRAB domain to the CSMD1 enhancer (fam3) in the neuronal precursor cell line – Lund human mesencephalic (LUHMES). Given that the expression of CSMD1 was not detectable in LUHMES cells we differentiated these cells into neurons. Differentiated neurons with CRISPRi of CSMD1 enhancer showed significantly higher expression of CSMD1 than control.

2022-06-30 | E-MTAB-8027 | biostudies-arrayexpress

Gene expression profiling (RNA-seq) experiment where CRISPRi guides were designed to target the human rs2650492 variant, the promoter of GAPDH, and a non-coding region near TUFM in HEK293t cells

Project description:CRISPRi guides were designed to target genomic regions of interest in HEK293t cells. Four guides were designed per region (see processed file). These regions were an enhancer located in the 3’UTR of SBK1 which harbors the obesity GWAS variant rs2650492, the promoter of GAPDH as a positive control, and a non-coding region near TUFM as a negative control. We also provide transcriptomes for Cas9 transfected cells only. Genes proximal to rs2650492 were assessed for differential expression. This experiment was performed with technical replicates where each replicate is a unique transfection and library preparation.

2021-07-14 | E-MTAB-10460 | biostudies-arrayexpress

A CRISPRi system for efficient and rapid gene knockdown in Caulobacter crescentus

Project description:CRISPR interference (CRISPRi) is a powerful new tool used in different organisms that provides a fast, specific, and reliable way to knockdown gene expression. Caulobacter crescentus is a well-studied model bacterium, and although a variety of genetic tools have been developed, it currently takes several weeks to delete or deplete individual genes, which significantly limits genetic studies. Here, we optimized a CRISPRi approach to specifically downregulate the expression of genes in C. crescentus. Although the Streptococcus pyogenes CRISPRi system commonly used in other organisms does not work efficiently in Caulobacter, we demonstrate that a catalytically-dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of Streptococcus thermophilus or from Streptococcus pasteurianus can each be effectively used in Caulobacter. We show that these CRISPRi systems can be used to rapidly and inducibly deplete ctrA or gcrA, two essential well-studied genes in Caulobacter, in either asynchronous or synchronized populations of cells. Additionally, we demonstrate the ability to multiplex CRISPRi-based gene knockdowns, opening new possibilities for systematic genetic interaction studies in Caulobacter.

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CRISPRi knockdown of essential genes in Pseudomonas aeruginosa

Project description:To understand molecular pathways responding to loss of essential proteins in the inner- or outer-membrane, we knocked down essential genes in the inner- and outer membrane using a CRISPRi approach in Pseudomonas aeruginosa. CRISPRi is an inducible system with which even essential genes can be targeted by a specifig sg-RNA. In this study, we investigated the transcriptomic changes upon loss of BamA, LptD and FtsH. Loss of BamA and LptD, both outer membrane proteins, resulted in strong upregulation of genes involved in LipidA modification and upregulation of several two component systems. Depletion of FtsH resulted in strong upregulation in genes responsible for amino acid biosynthesis and regulatory genes. Surprisngly, knockdown of bamA and lptD resulted in (mild) upregulation of H1-T6SS genes.

2020-12-21 | GSE159327 | GEO

Gene expression of T47D-MTVL human breast cancer cells upon H1X knock-down

Project description:Gene expression of T47D-MTVL human breast cancer cells expressing Dox-inducible shRNAs against histone H1X

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Expression data throughout reprogramming of MEF to iPS using a Dox-inducible promoter

Project description:6C secondary MEFs were treated with Dox in mES media to turn on the Oct4, Klf4, cMyc, Sox2. Total RNA was extracted at day 0 (no Dox), day2, 5, 8, 11, 16 and 21 (with Dox) and day 30 (Dox-independent secondary iPS). RNA from Parental MEFs and Primary-iPS cells were also extracted for reference. 1B secondary MEFs were Dox treated for 5-days followed by RNA extraction. Subsequently, a culture removed of Dox treatment for an additional 5days was also analyzed.

2010-07-01 | GSE21757 | GEO

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