Project description:Cas9 expressing eHAP cells were transduced with human sgRNA lentivirus library (Doench, 2016 Nature biotech, Addgene, #73178). The cells were selected with 1 µg/ml of puromycin for 2 days, and then used for ATAC-see at 5 and 7 days post-transduction, respectively. Cells in the G2 phase were specifically gated to sort th 5% of the cells with the highest and lowest ATAC-see signal intensity(“high” or “low” population) . The sgRNA enrichment in each population was analysed by high-throughput sequencing and computationally compared by using the MAGeCK program (Li et al., Genome Biology 2014).
Project description:This is an in vitro genome-wide CRISPR/cas9 screen in human glioblastoma stem cells, screening for genes essential for survival of these cells. These cells express cas9 and have been transfected with a guide RNA library causing gene knockouts. We will analyse the sequencing data for depletion of guide RNAs. In this particular study, we will do RNA sequencing to correlate CRISPR with expression levels in specific cancer cell subpopulations. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:We combined CRISPR genome editing with single-cell RNA sequencing to assess complex phenotypes in pooled cellular screens. Our method for CRISPR droplet sequencing (CROP-seq) comprises four key components: a gRNA vector that makes individual gRNAs detectable in single-cell transcriptomes, a high-throughput assay for single-cell RNA-seq, a computational pipeline for assigning single-cell transcriptomes to gRNAs, and a bioinformatic method for analyzing and interpreting gRNA-induced transcriptional profiles. CROP-seq allowed us to link gRNA expression to the associated transcriptome responses in thousands of single cells using a straightforward and broadly applicable screening workflow. Additional information are available from the CROP-seq website http://crop-seq.computational-epigenetics.org
Project description:Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as inflammation and cancer. Steps involved in cell migration include cell polarization, formation of membrane protrusions at the cell front side and adhesion disassembly at the rear side, and a general cytoskeletal rearrangement. However, there are cell-specific and context-specific molecular events acting in the process. Here, we show how to screen for genes and miRNAs involved in migration by the direct integration of a high-throughput gene editing method, the CRISPR-Cas9 knockout pool screening, and a well-established functional assay, the transwell migration assay. We applied this methodology to human fibroblasts migrating in the presence of a biochemical gradient. The results confirm known genes involved in migration, but also highlight new candidates, like the ectopically expressed family of olfactory receptors. This work establishes a methodological advancement in the use of CRISPR technology for functional screening, and represents a resource for candidate genes and miRNA playing a role in human fibroblast directional migration.
Project description:The role of nutrient signaling processes in the fate decision of CD8 are incompletely understood. By performing in vivo pooled CRISPR-Cas9 screening, we uncovered nutrient signaling processes underpinning the dynamics and heterogeneity of CD8 T cell fate decisions.
Project description:To assess the biological significances of p-hTERT in ALT cells, we used the CRISPR-Cas9 system targeting the hTERT gene and performed genome-wide CRISPR screening, which identified genes in the Fanconi anemia/BRCA pathway as synthetic lethal partners of hTERT.
Project description:Identifying new pathways that regulate mammalian regeneration is challenging due to the paucity of in vivo screening approaches. We employed pooled CRISPR knockout and activation screening in the regenerating liver to evaluate 164 chromatin regulatory proteins. Both screens identified imitation-SWI chromatin remodeling components Baz2a and Baz2b, not previously implicated in regeneration. In vivo sgRNA, siRNA, and knockout strategies against either paralog confirmed increased regeneration. Distinct BAZ2-specific bromodomain inhibitors GSK2801 and BAZ2-ICR resulted in accelerated liver healing after diverse injuries. Inhibitor treated mice also exhibited improved regeneration in an inflammatory bowel disease model, suggesting multi-tissue applicability. Transcriptomics on regenerating livers showed increases in ribosomal and cell cycle mRNAs. Surprisingly, CRISPRa screening to define mechanisms showed that overproducing ribosomal proteins was sufficient to drive regeneration, while Rpl24 haploinsufficiency was rate limiting for BAZ2 inhibition mediated regeneration. The discovery of regenerative roles for imitation-SWI components provides immediate strategies to enhance tissue repair.