Project description:In this study, we investigated the transcriptional responses induced by in vitro–transcribed (IVT) sgRNAs in a CRISPR interference (CRISPRi) system. A total of 906 sgRNAs were generated using a commercially available IVT workflow and delivered to HEK293T cells stably expressing dCas9-KRAB-MECP2. RNA sequencing was performed to profile genome-wide transcriptional changes following sgRNA transfection. Compared with mock-transfected controls, sgRNA-transfected cells exhibited a strong and consistent upregulation of interferon-stimulated genes (ISGs), with prominent enrichment of interferon-α response pathways across the majority of sgRNAs. While most sgRNAs induced robust interferon signaling, a small subset showed a more limited transcriptional response. IFIT1 and IFIT2, two canonical markers of interferon activation, were consistently upregulated and were selected as representative genes for downstream validation. This dataset provides a systematic resource for assessing innate immune activation triggered by IVT sgRNAs in CRISPRi-based perturbation experiments.
Project description:To identify genes driving encephalitogenic CD4+ T cell migration into the CNS, we performed a genome-wide CRISPR screen and a subsequent validation screen For the genome-wide CRISPR screen, up to 4 sgRNA per gene and 800 non targeted controls were included, for a total of 87690 individual sgRNAs, and for the validation screen, up to 6 sgRNA per gene and 241 non targeted controls for a total of 12000 individual sgRNAs
Project description:Solar ultraviolet (UV) exposure activates Toll-Like Receptor 4 (TLR4), initiating inflammatory signaling pathways relevant to human skin carcinogenesis. Using transcriptomic (Nanostring-based) and proteomic (reverse phase protein array-based) analysis we demonstrate that genetic deletion of epidermal Tlr4 mimics the photoprotective impact of pharmacological antagonism using topical resatorvid (RES), an effect not observed in transgenic mice harboring systemic deletion of Tlr4. These findings suggest that epidermal TLR4 represents a promising target for skin cancer photochemoprevention.
Project description:Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation in vivo, via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters gene expression, lipid metabolic pathways, and membrane lipid composition, which are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.
Project description:CT26 cells expressing lentiviral Cas9 and sgRNAs targeting either control or Gna13 were transplanted into immunocompetent BALB/c mice. Tumors were harvested and processed for RNA-seq
Project description:To gain insights into how EBV latency is maintained, we performed a human genome-wide CRISPR screen in latently EBV-infected Burkitt lymphoma B-cells. Our analyses identified a network of host factors that repress EBV lytic reactivation, centered on the transcription factor MYC and including cohesins, FACT, STAGA and Mediator. RNAseq was used to identify host and viral transcriptome changes in P3HR-1 Burkitt lymphoma cells expressing control, smc1a, supt16h, med12, or tada2b sgRNAs. RNAseq was used to identify host and viral transcriptome changes in Akata EBV+ burkitt lymphoma cells expressing control or myc sgRNAs.