Effects of IL2RA non-coding and coding sequence edits on IL2RA expression in primary human T cells.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE88960 | GEO | 2017/07/30
SECONDARY ACCESSION(S): PRJNA349190
REPOSITORIES: GEO
Similar Datasets
Project description:Adenosine deaminases (ADARs) are RNA binding proteins that bind to double stranded RNA and convert adenosine to inosine. Editing creates multiple isoforms of neurotransmitter receptors, including AMPA-subtype Glutamate channels such as Gria2 that is edited to introduce a Q to R amino acid change. Adar2 knock out mice die of seizures shortly after birth, but if the Gria2 Q/R editing site is mutated to mimic the edited version then the animals are viable. We performed RNA-Seq on the frontal cortices of Adar2-/- Gria2R/R mice and their Adar2+/+ Gria2R/R littermates, quantifying overall gene expression, splicing, and A to I editing in a transcriptome-wide fashion. We found 56 editing sites whose level of editing was significantly diminished in the Adar2 deficient animals. The majority of Adar2 responsive editing sites were in the coding regions of genes. Interestingly, other than Adar2 expression, there were only two additional statistically significant differentially expressed genes, Flnb and Cdh13. There were also only three exons that showed statistically significant differences in expression levels between the two genotypes. This work illustrates that ADAR2 is important in site-specific changes of protein coding sequences but has only modest effects on gene expression or splicing in vivo. 6 ADAR2 Knock out frontal cortices and 6 litter mate control frontal cortices from male mice
2016-07-03 | E-GEOD-70588 | biostudies-arrayexpress
Project description:Adenosine deaminases (ADARs) are RNA binding proteins that bind to double stranded RNA and convert adenosine to inosine. Editing creates multiple isoforms of neurotransmitter receptors, including AMPA-subtype Glutamate channels such as Gria2 that is edited to introduce a Q to R amino acid change. Adar2 knock out mice die of seizures shortly after birth, but if the Gria2 Q/R editing site is mutated to mimic the edited version then the animals are viable. We performed RNA-Seq on the frontal cortices of Adar2-/- Gria2R/R mice and their Adar2+/+ Gria2R/R littermates, quantifying overall gene expression, splicing, and A to I editing in a transcriptome-wide fashion. We found 56 editing sites whose level of editing was significantly diminished in the Adar2 deficient animals. The majority of Adar2 responsive editing sites were in the coding regions of genes. Interestingly, other than Adar2 expression, there were only two additional statistically significant differentially expressed genes, Flnb and Cdh13. There were also only three exons that showed statistically significant differences in expression levels between the two genotypes. This work illustrates that ADAR2 is important in site-specific changes of protein coding sequences but has only modest effects on gene expression or splicing in vivo.
2016-01-01 | GSE70588 | GEO
Project description:We report transcriptome wide edits comparison between split-engineered base editors and intact base editors. Our results show that, split-engineered base editors show backgound levels of unique C>U edits when compared to intact base editors.
2021-10-18 | GSE181109 | GEO
Project description:We previously performed a tiling CRISPR activation (CRISPRa) screen in Jurkat T cells to discover enhancers controlling IL2RA expression. This screen identified six regions where recruitment of dCas9-VP64 was sufficient to drive increased expression of IL2RA. We named these putative enhancers CRISPRa Responsive Elements (CaREs). To examine transcriptome-wide consequences of dCas9-VP64 recruitment to IL2RA CaREs, we performed RNA-Seq on HuT78 cells stably expressing dCas9-VP64 and gRNAs targeting the IL2RA TSS, CaRE3, or CaRE4, or stimulated with anti-CD3/CD28 antibodies.
2017-04-26 | GSE98177 | GEO
Project description:A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to measure Cas9 edit outcomes at single-cell resolution. Here we present Superb-seq, a new technology for joint measurement of on-target and off-target Cas9 edits and transcriptomes by single-cell RNA sequencing. In contrast to Perturb-seq methods that read each cell’s guide RNA, Superb-seq directly reads Cas9 edits by leveraging T7 in situ transcription. We performed Superb-seq on 9,500 K562 cells, targeting four chromatin remodeler genes with seven guide RNAs. Superb-seq identified 11,891 edit events in 6,230 edited cells at all seven on-target sites and at 36 off-target sites. One notable off-target edit fell within the first intron of the deubiquitinase gene USP9X, which decreased its expression and perturbed the expression of downstream genes. Superb-seq uses off-the-shelf kits, standard equipment, and requires no virus, which will enable CRISPR screens in virus-intolerant cell types and functional characterization of off-target events.
2025-02-25 | GSE284207 | GEO
Project description:A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to characterize editing outcomes at single-cell resolution. We present "Superb-seq", a new scRNA-seq method that measures on-target and off-target genome edits and associated transcriptomes. In contrast to Perturb-seq that captures single-cell guide information, Superb-seq directly captures single-cell edit profiles by T7 in situ transcription. We demonstrate and validate this method through two Superb-seq experiments in 30,000 cells of three cell types, amplicon sequencing (rhAmpSeq), and bulk RNA-seq. Superb-seq uses off-the-shelf kits, standard laboratory equipment, and requires no virus, which will improve CRISPR screens in diverse tissues and functional characterization of CRISPR therapeutics.
2025-12-17 | GSE313955 | GEO
Project description:A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to characterize editing outcomes at single-cell resolution. We present "Superb-seq", a new scRNA-seq method that measures on-target and off-target genome edits and associated transcriptomes. In contrast to Perturb-seq that captures single-cell guide information, Superb-seq directly captures single-cell edit profiles by T7 in situ transcription. We demonstrate and validate this method through two Superb-seq experiments in 30,000 cells of three cell types, amplicon sequencing (rhAmpSeq), and bulk RNA-seq. Superb-seq uses off-the-shelf kits, standard laboratory equipment, and requires no virus, which will improve CRISPR screens in diverse tissues and functional characterization of CRISPR therapeutics.
2025-12-17 | GSE313958 | GEO
Project description:Adenosine deaminases bind double stranded RNA and convert adenosine to inosine. Editing creates multiple isoforms of neurotransmitter receptors, such as with Gria2. Adar2 KO mice die of seizures shortly after birth, but if the Gria2 Q/R editing site is mutated to mimic the edited version then the animals are viable. We performed RNA-Seq on frontal cortices of Adar2(-/-) Gria2(R/R) mice and littermates. We found 56 editing sites with significantly diminished editing levels in Adar2 deficient animals with the majority in coding regions. Only two genes and 3 exons showed statistically significant differences in expression levels. This work illustrates that ADAR2 is important in site-specific changes of protein coding sequences but has relatively modest effects on gene expression and splicing in the adult mouse frontal cortex.
| S-EPMC4829308 | biostudies-literature
Project description:We performed tiling CRISPR activation (CRISPRa) screens in Jurkat T cells to discover enhancers controlling IL2RA and CD69 expression. For each target gene, we constructed a pooled lentiviral library of sgRNAs that targeted sites at all Cas9 PAMs throughout the window starting 100 kb upstream of the transcription start site, extending through the gene body, and 25 kb downstream. A separate library was constructed for each gene, and a separate screen was performed for each gene. For each gene, Jurkat cells stably expressing the dCas9-VP64 transcriptional activator were transduced with the lentiviral sgRNA library. Cells were stained for the protein of interested and sorted by flow cytometry into four bins of expression. Distribution of sgRNAs in each of the sorted populations was compared to the unsorted population. Regions where dCas9-VP64 recruitment was sufficient to drive target gene expression were identified as putative enhancers. The hg19 coordinates of the CD69 library window were chr12:9,880,082-10,013,497. The library contained 2,244 negative control sgRNAs taken from the CRISPRi/a libraries described in Gilbert et al., Cell 150, 647-661 (2014). The hg19 coordinates of the IL2RA library window were chr10:6,027,657- 6,204,333. The library contained 2,244 negative control sgRNAs taken from the CRISPRi/a libraries described in Gilbert et al., Cell 150, 647-661 (2014).
2017-04-26 | GSE98178 | GEO
Project description:Effects of IL2RA non-coding and coding sequence edits on IL2RA expression in primary human T cells.
| PRJNA349190 | ENA