Similar Datasets
Project description:Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy in several cancer types. However, only a fraction of patients will respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMM) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types. KMT2D encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in patients with cancer. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. In addition, Kmt2d-mutant cells exhibited increased protein turnover and IFNγ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human were characterized by increased immune infiltration. These data demonstrate that Kmt2d deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, and also highlight the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment. SIGNIFICANCE: ICB is ineffective in the majority of patients. Through direct in vivo CRISPR mutagenesis screening in GEMMs of cancer, we find Kmt2d deficiency sensitizes tumors to ICB. Considering the prevalence of KMT2D mutations, this finding potentially has broad implications for patient stratification and clinical decision-making.This article is highlighted in the In This Issue feature, p. 1775.
| S-EPMC7710536 | biostudies-literature
Project description:Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy across multiple cancer types. However, only a fraction of patients respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMMs) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types. Kmt2d encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in cancer patients. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. Additionally, Kmt2d-deficient cells exhibit increased protein turnover and IFN-γ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human are characterized by increased immune infiltration. These data demonstrate that KMT2D deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, highlighting the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment.
2020-11-18 | GSE151226 | GEO
Project description:Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy across multiple cancer types. However, only a fraction of patients respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMMs) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types. Kmt2d encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in cancer patients. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. Additionally, Kmt2d-deficient cells exhibit increased protein turnover and IFN-γ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human are characterized by increased immune infiltration. These data demonstrate that KMT2D deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, highlighting the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment.
2020-11-18 | GSE151225 | GEO
Project description:CRISPR-GEMM pooled mutagenic screening identifies KMT2D as a major modulator of immune checkpoint blockade
| PRJNA635149 | ENA
Project description:CRISPR-GEMM pooled mutagenic screening identifies KMT2D as a major modulator of immune checkpoint blockade (RNA-Seq)
| PRJNA635150 | ENA
Project description:CRISPR-GEMM pooled mutagenic screening identifies KMT2D as a major modulator of immune checkpoint blockade (ATAC-Seq)
| PRJNA635151 | ENA
Project description:Components of the type II CRISPR-Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR-Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening.
| S-EPMC4992892 | biostudies-other
Project description:CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.
| S-EPMC5334791 | biostudies-literature
Project description:Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale.
| S-EPMC6258485 | biostudies-literature
Project description:Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3' untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments.
| S-EPMC6561216 | biostudies-literature