CRISPR/Cas9 in vivo screens in patients’ leukemias growing in mice
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ABSTRACT:
PROVIDER: PRJNA580244 | ENA |
REPOSITORIES: ENA
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Project description:Functional genomic screens were mostly performed in cell lines and mouse models so far. To approximate the clinical situation, we established customized CRISPR/Cas9 screens in patient-derived xenograft (PDX) leukemia models in vivo. Ninety-two surface molecules were tested; sgRNAs against CXCR4 and ITGB1 dropped out and single knockouts validated their essential function. Our method allows determining patient-specific tumor dependencies in vivo which might facilitate personalized medicine of cancer in the future.
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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/
2015-02-03 | E-ERAD-335 | biostudies-arrayexpress
Project description:Acute leukemias represent deadly malignancies which require better treatment. As challenge, treatment is counteracted by a microenvironment protecting dormant leukemia stem cells. To identify responsible surface proteins, we performed deep proteome profiling on minute numbers of dormant patient-derived xenograft (PDX) leukemia stem cells isolated from mice. Candidates were functionally screened by establishing a comprehensive CRISPR-Cas9 pipeline in PDX models in vivo. A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as essential vulnerability required for survival and growth of different types of acute leukemias in vivo and reconstitution assays in PDX models proved the relevance of its sheddase activity. Of translational importance, molecular or pharmacological targeting of ADAM10 reduced PDX leukemia burden, cell homing to the murine bone marrow and stem cell frequency, and increased leukemia response to conventional chemotherapy in vivo. These findings identify ADAM10 as attractive therapeutic target for future treatment of acute leukemias.
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Project description:H1-HeLa cells were stably transduced with lentiCas9-Blast (Addgene, Plasmid #52962) and subsequently selected using blasticidin to generate constitutively expressing Cas9 H1-HeLa cells. A single Cas9-expressing H1-HeLa clone was then transduced with lentivirus without a selection marker to stably express CDHR3 C529Y (H1-HeLa+CDHR3). A single CDHR3-expressing H1-HeLa clone was then chosen based on RT-qPCR of CHDR3 expression and RV-C15 RNA levels for mutagenesis. 300 million of the H1-HeLa cells constitutively expressing CDHR3 and Cas9 were transduced with the lentiGuide-Puro from the GeCKO v2 library at a MOI of 0.3. Cells were selected using puromycin and heterogeneous H1-HeLa knockout cell populations were subsequently pooled together. The CRISPR genetic screens were started 10 days post transduction. >1000-fold coverage of mutagenized cells (libraries A and B) was infected with either RV-C15 (MOI=1 PFU/cell) or EV-D68 Missouri (MOI=1 PFU/cell). RV-C15 infection was repeated for an additional round at 6 days post-infection. As soon as appearance of visibly viable colonies was observed, populations of virus-resistant cells were pooled and harvested. Uninfected starting populations of mutagenized cells were used as the unselected reference. Total genomic DNA from both virus-resistant and uninfected cells was respectively extracted using QIAamp DNA Mini Kit (Qiagen). The inserted guide RNA sequences were retrieved from the genomic DNA by PCR amplification. The PCR products were then purified and subjected to NextSeq platform (Illumina) next-generation sequencing.
2019-07-10 | E-MTAB-8125 | biostudies-arrayexpress
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.
2016-02-15 | E-GEOD-70038 | biostudies-arrayexpress
Project description:CRISPR screens have become standard gene discovery platforms in various contexts, including cancer. Yet, commonly available CRISPR-Cas9 tools are increasingly recognized as unfit for in vivo investigations in immunocompetent contexts, due to the broad immunogenicity of bacterial nucleases and reporters. Here, we show how CRISPR screens with tumor grafts are systematically jeopardized by immunoediting of Cas9-expressing cells in immunocompetent hosts, resulting in iatrogenic clonal dropouts and ultimately compromising target identification.
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Project description:CRISPR/Cas9 screens identified unexpected hits in human AML cell lines whose loss of function causes resistance to SHP2 inhibition
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Project description:CRISPR screens have become standard gene discovery platforms in various contexts, including cancer. Yet, commonly available CRISPR-Cas9 tools are increasingly recognized as unfit for in vivo investigations in immunocompetent contexts, due to the broad immunogenicity of bacterial nucleases and reporters. Here, we show how CRISPR screens with tumor grafts are systematically jeopardized by immunoediting of Cas9-expressing cells in immunocompetent hosts, resulting in iatrogenic clonal dropouts and ultimately compromising target identification.
2025-10-29 | GSE268045 | GEO
Project description:CRISPR screens have become standard gene discovery platforms in various contexts, including cancer. Yet, commonly available CRISPR-Cas9 tools are increasingly recognized as unfit for in vivo investigations in immunocompetent contexts, due to the broad immunogenicity of bacterial nucleases and reporters. Here, we show how CRISPR screens with tumor grafts are systematically jeopardized by immunoediting of Cas9-expressing cells in immunocompetent hosts, resulting in iatrogenic clonal dropouts and ultimately compromising target identification.
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