Project description:To identify new therapeutic targets for Glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 "knockout" (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Tyr15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, likely as a result of oncogenic signaling, causing GBM-specific lethality. A whole-genome CRISPR-Cas9 knockout screens targeting over 18,000 genes using the all-in-one LV-sgRNA:Cas9 platform system were performed using a âshot gunâ approach by transducing 2 GBM patient-derived isolates and 2 human neural stem cell isolates with the pool library (2 biological replicates), and cultures were outgrown for ~3 weeks. The end time point of each screen was compared to day 0 in order to determine which sgRNAs were overrepresented or underrepresented in the population.
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: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:Acute myeloid leukaemia (AML) continues to have a very high mortality and its treatment hasnot changed for more than 20 years.Here, we propose to apply genomic-wide screens to directly identify therapeutic vulnerabilitiesof AML using AML cells lines with pre-defined somatic mutations. Our studies will be basedon genome-wide CRISPR/CAs9 gRNA screens and will be used to identify drivers of drugresistance to standard chemotherapies.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:Genome-wide CRISPR-Cas9 knockout screens were performed in dual-fluorescent frameshifting reporter cell lines to identify human host factors for SARS-CoV-2 programmed ribosomal frameshfiting.
Project description:CRISPR knockout screens have accelerated the discovery of important cancer genetic dependencies. However, traditional CRISPR-Cas9 screens are limited in their ability to assay the function of redundant or duplicated genes. Paralogs in multi-gene families constitute two-thirds of the protein-coding genome, so this blind spot is the rule, not the exception. To overcome the limitations of single gene CRISPR knockout screens, we developed paired guide RNAs for Paralog gENetic interaction mapping (pgPEN), a pooled CRISPR/Cas9 approach which targets over a thousand duplicated human paralogs in single knockout and double knockout configurations. We applied pgPEN to two cell lineages and discovered that over 10% of human paralogs exhibit synthetic lethality in at least one cellular context. We recovered known synthetic lethal paralogs such as MAP2K1/MAP2K2, important drug targets such as CDK4/CDK6, and numerous other synthetic lethal pairs such as CCNL1/CCNL2. In addition, we identified ten tumor suppressive paralog pairs whose compound loss promotes cell growth. These findings identify a large number of previously unidentified essential gene families and nominate new druggable targets for oncology drug discovery.
