Project description:A genome-wide CRISPR-Cas9 knockout screen was performed in the breast cancer cell lines T47D, MCF7, and CAMA-1 to identify genes modulating sensitivity and resistance to capivasertib, a selective AKT inhibitor. Cells were transduced with a CRISPR library, followed by treatment with capivasertib or vehicle control (DMSO). Guide RNA (sgRNA) enrichment and depletion were assessed via next-generation sequencing to determine gene-level effects on cell viability and drug response. Gene-level data are provided for the initial plasmid library, baseline, DMSO-treated controls, and post-capivasertib treatment across replicates.
Project description:Increased or decreased B16 cell sensitivity to activated cytotoxic T lymphocytes.To identify tumor cell-intrinsic genes essential for antigen-specific CD8+ T cell killing, we utilized the B16.SIY melanoma cell line which expressed the model antigen SIY (SIYRYYGL) co-expressed with DsRed, that can be recognized by 2C CD8+ TCR transgenic (Tg) T cells. B16.SIY cells were transduced and selected to stably expressed a Cas9 gene, and then transduced with a genome-scale CRISPR gRNA library.Transduced cells were cultured for 8 days to provide time for gene disruption to occur, then were co-cultured with pre-activated 2C CD8+ T cells at a 2:1 Effector:Target ratio for 16 hours. By performing deep sequencing, we examined the sgRNA library representation in tumor cells with or without T cell co-incubation by MAGeCK analysis.
Project description:To identify genes involved in macrophage differentiation, a genome-wide CRISPR knockout library in macrophage progenitors was differentiated into macrophages. Samples were collected from the progenitor library at day 0, from a library maintained as progenitors for seven days, and from macrophages after seven days of differentiation.
Project description:To elucidate the early regulators of inflammation during Mycobacterium tuberculosis infection, we performed a genome-wide CRISPR knockout screen in macrophages to identify genes that influence the induction of TNF and iNOS upon infection. A genome-wide knockout library in murine macrophages was infected with Mycobacterium tuberculosis and 24 hours post infection cells were FACS sorted based on TNF and iNOS expression. Samples were collected from the unsorted library, as well as two sorted populations: TNF positive and iNOS negative, and TNF positive and iNOS positive.
Project description:(i) RNA-seq of M. tuberculosis H37Rv cultures grown in the presence of BVL3572S for 6 h compared to untreated controls. (ii) Genome-wide M. tuberculosis H37Rv CRISPRi libraries were grown in the presence of ATc for 5 days prior to drug exposure. Cultures were treated with sub-MIC BVL3572S concentrations without amino acid or in the presence of L-His, L-Ala, or both. After 14 days outgrowth, genomic DNA were harvested from cultures for which BVL3572S concentrations were partially growth inhibitory and from untreated controls. Abundance of sgRNA was analyzed by deep sequencing. (iii) Genome-wide M. tuberculosis H37Rv transposon mutant librairies were treated with BVL3572S or untreated for 15 days followed by a 7-day antibiotic-free outgrowth. Genomic DNA were extracted, indexed, and sequenced.
Project description:To uncover, in an unbiased fashion, which elements of the 18 kb translocated region control EVI1 transcription, we devised a CRISPR/Cas9-based enhancer scanning approach. We considered all possible sgRNA target sites containing a canonical Cas9 PAM site (NGG) on both strands of the minimal 18 kb translocated region. Deep-sequencing libraries were generated by PCR amplification of sgRNA guide strands using primers that tag the product with standard Illumina adapters and a 4 bp sample barcode in a 2 step-PCR protocol.
Project description:To identify genes driver resistance of apatinib on human umbilical vein endothelial cells (HUVECs). The CRISPR-Pool SAM human library, containing 70290 sgRNAs targeting 23430 genes were constructed in MS2-P65-HSF1. After the cells were treated with apatinib for another week and their genomic DNA was extracted. The sgRNA fragments were amplified by PCR. After passing quality control, 20–50 ng of DNA fragments are used to construct a high-throughput sequencing library for the Illumina platform through steps such as End Repair, 5' Phosphorylation and dA-Tailing, Adapter Ligation, Clean Up, and PCR Enrichment and Clean Up.
Project description:Unbiased forward genetic screens to identify host factors for DENV1 and JEV in 293FT cells. Unbiased forward genetic screens to identify host factors for HCoV-229E in Huh7.5.1 cells.
Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.