IPSC RNA-seq upon CRISPRn or CRISPRi manipulation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Max Horlbeck
PROVIDER: E-GEOD-76980 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3 to 4 sgRNAs binding to the proximal promoters suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system. We used microarray to assay the gene expression changes after transfection of dCas9VP160 with the different sgRNAs
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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).
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