Project description:The exact functional roles of most of the dysregulated metabolic genes in tumorigenicity are still unclear. We report the application of CRISPR/Cas9 knockout screen technology in hepatocellular carcinoma cells. By an in vivo CRISPR/Cas9 knockout screen that targets 1,121 differentially expressed metabolic genes in HCC, we identified 67 metabolic genes as oncogenic candidates for HCC.
Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library was performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
Project description:The coordinated activation of DNA replication origins is important for efficient DNA synthesis and genome stability. S-phase cyclin dependent kinases (CDKs) together with CDC7 kinase, are essential to origin activation by converting the pre-replicative complex into a fully active helicase. To identify genes that tune DNA replication, we performed a chemo-genetic genome-wide CRISPR-KO screen with cells challenged with the CDC7 inhibitor XL413. By developing a methodology based on genetic coessentiality to functionally cluster the hits, we uncover the transcriptional CDK8/CCNC kinase in a cluster with replication initiation factors. We find that CDK8 depletion further reduces the rate of DNA synthesis imposed by CDC7 inhibitors. DNA fibre experiments provide compelling evidence that CDK8 and CDC7 cooperate in origin activation. Suppression of DNA synthesis by CDK8 inhibition requires the binding of CDK8/CCNC to the MDM2 Binding Protein (MTBP) and we show that CDC7 and CDK8 associate to the MCM complex and individually contribute to the phosphorylation of the replicative helicase. Thus, this work identifies CDK8 as the latest kinase directly involved in origin activation in human cells. Pooled genome wide CRISPR/Cas9 screen outline Cell line: MCF10A cells were stably transduced with EditR Lentiviral Cas9 nuclease (hEF1a promoter, Blasticidin resistance marker, Dharmacon) and subsequently selected using blasticidin to generate constitutively expressing Cas9 MCF10A cells. A single clone was isolated (MCF10A EditR cell line) and used for pooled genome-wide CRISPR/Cas9 screens. CRISPR pooled lentiviral sgRNA library: A plasmid based CRISPR pooled lentiviral sgRNA library that can be used in a modular format was purchased from CELLECTA (CELLECTA: CRISPR Human Genome Knockout Library, Module 1 (200 µg plasmid), #sgRNA: 52,549, KOHGW-M1-P; Module 2 (200 µg plasmid), #sgRNA: 49,461, KOHGW-M2-P; and Module 3 (200 µg plasmid), #sgRNA: 48,578, KOHGW-M3-P). Each module covers approximately 6,300 genes and each gene is targeted by 8x sgRNAs (~50,000 sgRNAs per module), in total targeting >19,000 protein coding genes.
Project description:A validation experiment performed on HEK293 cell lines after genome editing. The design contains three duplicate runs consisted of: HEK293 wild type cell line HEK293 with MIR484 gene knockdown using CRISPR-Cas9 HEK293 with MIR185 gene knockout using CRISPR-Cas9
Project description:A secondary custom CRISPR-Cas9 screen, encompassing the 132 genes identified in the genome-wide TP53 knockout screen along with additional 93 genes serving as positive and negative controls.
Project description:Haploid human embryonic stem cells harboring TP53 or MLH1 knockout (KO) were subjected to a genome-wide CRISPR-Cas9 knockout screen to identify synthetic lethal interactions associated with the mentioned genes.
Project description:This study describes a genome-wide CRISPR-Cas9 knockout (GeCKO) screen performed in two porcine cell lines, PK15 and IPEC-J2. A custom-designed porcine GeCKO (pGeCKO) library targeting protein-coding genes, lncRNAs, and miRNAs was used. Genomic DNA was harvested from GeCKO cell populations at four timepoints between 3-26 days post-transduction in each cell line. Amplicon sequencing libraries were generated from guide RNA regions using custom primers with Illumina adapter sequences, and sequenced on the NovaSeq 6000 platform (PE150). This dataset enables quantification of guide RNA abundance over time and identification of essential genes in pig cells, supporting research in comparative genomics, functional genomics, and translational animal models.
Project description:Metastasis is the main cause of cancer-related deaths, yet the underlying mechanisms remain elusive. Using clear cell renal cell carcinoma (ccRCC), a tumor type with frequent lung metastases, we conducted an in vivo genome-wide CRISPR-Cas9 screen and identified HLF as a potent suppressor of lung metastasis. While HLF translocation is known as an oncogenic event in leukemia, its role in solid cancers remains unclear. Our study revealed that HLF depletion enhanced ccRCC migration and lung metastasis, whereas HLF overexpression abrogated these effects. This finding extended to multiple solid tumor types. In ccRCC patients, HLF expression was reduced at metastatic sites and associated with epigenetic silencing. HLF levels negatively correlated with migration potential in collagen. Mechanistically, HLF regulated leupaxin expression, affecting the integration of collagen stiffness and the actin cytoskeleton through paxillin, thereby repressing cancer cell migration and lung metastasis. These data indicate that HLF influences lung metastasis through cell-collagen interactions in solid tumors.
2025-04-22 | GSE272563 | GEO
Project description:SOX11 knockout by CRISPR/Cas9 in SKNBE2 cell line