916 results match your criteria crispr screens

Gene fusions create partner and collateral dependencies essential to cancer cell survival.

Cancer Res 2021 Jun 7. Epub 2021 Jun 7.

Department of Medical Oncology, Dana-Farber Cancer Institute

Gene fusions frequently result from rearrangements in cancer genomes. In many instances, gene fusions play an important role in oncogenesis; in other instances, they are thought to be passenger events. Although regulatory element rearrangements and copy number alterations resulting from these structural variants (SV) are known to lead to transcriptional dysregulation across cancers, the extent to which these events result in functional dependencies with an impact on cancer cell survival is variable. Read More

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RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library.

CRISPR J 2021 Jun 4. Epub 2021 Jun 4.

Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham Research Campus, Cambridge, United Kingdom.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing system has been broadly adopted for high-throughput genetic screens. However, the application of genome-wide single guide RNA (sgRNA) libraries can be challenging. We generated a custom sgRNA library, an order of magnitude smaller than genome-wide alternatives, to facilitate the genetic screening of RNA binding proteins (RBPs). Read More

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A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes.

Genome Res 2021 Jun 4. Epub 2021 Jun 4.

University of Oxford, MRC Weatherall Institute of Molecular Medicine

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the lysine methyltransferase 2A (KMT2A) produce KMT2A fusion proteins such as KMT2A-AFF1, causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). Read More

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Short and long sleeping mutants reveal links between sleep and macroautophagy.

Elife 2021 Jun 4;10. Epub 2021 Jun 4.

Chronobiology and Sleep Institute, Perelman Medical School of University of Pennsylvania, Philadelphia, United States.

Sleep is a conserved and essential behavior, but its mechanistic and functional underpinnings remain poorly defined. Through unbiased genetic screening in , we discovered a novel short-sleep mutant we named . Positional cloning and subsequent complementation, CRISPR/Cas9 knock-out, and RNAi studies identified Argus as a transmembrane protein that acts in adult peptidergic neurons to regulate sleep. Read More

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Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

Res Sq 2021 May 27. Epub 2021 May 27.

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. Read More

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Applications of Deep Mutational Scanning in Virology.

Viruses 2021 05 28;13(6). Epub 2021 May 28.

College of Medicine and Public Health, Flinders University, Bedford Park, SA 5042, Australia.

Several recently developed high-throughput techniques have changed the field of molecular virology. For example, proteomics studies reveal complete interactomes of a viral protein, genome-wide CRISPR knockout and activation screens probe the importance of every single human gene in aiding or fighting a virus, and ChIP-seq experiments reveal genome-wide epigenetic changes in response to infection. Deep mutational scanning is a relatively novel form of protein science which allows the in-depth functional analysis of every nucleotide within a viral gene or genome, revealing regions of importance, flexibility, and mutational potential. Read More

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Ultra-high-throughput single-cell RNA sequencing and perturbation screening with combinatorial fluidic indexing.

Nat Methods 2021 Jun 31;18(6):635-642. Epub 2021 May 31.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable cost-effective single-cell sequencing for millions of individual cells, we developed 'single-cell combinatorial fluidic indexing' (scifi). The scifi-RNA-seq assay combines one-step combinatorial preindexing of entire transcriptomes inside permeabilized cells with subsequent single-cell RNA-seq using microfluidics. Read More

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Pooled CRISPR screening in pancreatic cancer cells implicates co-repressor complexes as a cause of multiple drug resistance via regulation of epithelial-to-mesenchymal transition.

BMC Cancer 2021 May 29;21(1):632. Epub 2021 May 29.

HudsonAlpha Institute for Biotechnology, Huntsville, AL, 35806, USA.

Background: Pancreatic ductal adenocarcinoma (PDAC) patients suffer poor outcomes, including a five-year survival of below 10%. Poor outcomes result in part from therapeutic resistance that limits the impact of cytotoxic first-line therapy. Novel therapeutic approaches are needed, but currently no targeted therapies exist to treat PDAC. Read More

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CRISPR Screens in Toxicology Research: An Overview.

Curr Protoc 2021 May;1(5):e136

Department of Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida.

The use of genome editing tools is expanding our understanding of various human diseases by providing insight into gene-disease interactions. Despite the recognized role of toxicants in the development of human health issues and conditions, there is currently limited characterization of their mechanisms of action, and the application of CRISPR-based genome editing to the study of toxicants could help in the identification of novel gene-environment interactions. CRISPR-based functional screens enable identification of cellular mechanisms fundamental for response and susceptibility to a given toxicant. Read More

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Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses.

bioRxiv 2021 May 21. Epub 2021 May 21.

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. Read More

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Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis.

Nat Neurosci 2021 May 24. Epub 2021 May 24.

Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA.

Single-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type-specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Read More

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In vivo genome-wide CRISPR screen reveals breast cancer vulnerabilities and synergistic mTOR/Hippo targeted combination therapy.

Nat Commun 2021 05 24;12(1):3055. Epub 2021 May 24.

Department of Medicine, McGill University Health Center, Cancer Research Program, Montreal, QC, Canada.

Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Read More

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Zmat3 splices together p53-dependent tumor suppression.

Mol Cell Oncol 2021 29;8(3):1898523. Epub 2021 Apr 29.

Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA.

The tumor protein p53 (TP53, best known as p53) transcription factor is a critical tumor suppressor, but those p53-inducible genes most important for tumor suppression have remained unclear. Using unbiased RNA interference and CRISPR (Clustered Regularly Interspersed Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) screens, genetically engineered mouse models, human cancer genome analysis, and integrative eCLIP-sequencing and RNA-sequencing analyses, we reveal a new branch of p53-mediated tumor suppression involving the RNA splicing regulator Zinc finger Matrin-type 3, Zmat3. Read More

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Discovering antiviral restriction factors and pathways using genetic screens.

J Gen Virol 2021 May;102(5)

Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews, KY16 9ST, UK.

Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however, untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy, several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Read More

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A method for benchmarking genetic screens reveals a predominant mitochondrial bias.

Mol Syst Biol 2021 May;17(5):e10013

Department of Computer Science and Engineering, University of Minnesota - Twin Cities, Minneapolis, MN, USA.

We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene-pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. Read More

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High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer.

Nat Commun 2021 05 20;12(1):2969. Epub 2021 May 20.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

Chromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Read More

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Protein neddylation as a therapeutic target in pulmonary and extrapulmonary small cell carcinomas.

Genes Dev 2021 Jun 20;35(11-12):870-887. Epub 2021 May 20.

Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

Small cell lung carcinoma (SCLC) is among the most lethal of all solid tumor malignancies. In an effort to identify novel therapeutic approaches for this recalcitrant cancer type, we applied genome-scale CRISPR/Cas9 inactivation screens to cell lines that we derived from a murine model of SCLC. SCLC cells were particularly sensitive to the deletion of NEDD8 and other neddylation pathway genes. Read More

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-Deficient Zebrafish Reproduce Neurological and Inflammatory Symptoms of Niemann-Pick Type C Disease.

Front Cell Neurosci 2021 27;15:647860. Epub 2021 Apr 27.

Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disease that is caused by a mutation of the or gene, in which un-esterified cholesterol and sphingolipids accumulate mainly in the liver, spleen, and brain. Abnormal lysosomal storage leads to cell damage, neurological problems, and premature death. The time of onset and severity of symptoms of NPC disease are highly variable. Read More

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CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion.

Nat Commun 2021 05 11;12(1):2625. Epub 2021 May 11.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.

CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Read More

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In vivo CRISPR screening for novel noncoding RNA functional targets in glioblastoma models.

J Neurosci Res 2021 May 10. Epub 2021 May 10.

Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

CRISPR (clustered regularly interspaced short palindromic repeat)-based genetic screens offer unbiased and powerful tools for systematic and specific evaluation of phenotypes associated with specific target genes. CRISPR screens have been utilized heavily in vitro to identify functional coding and noncoding genes in a large number of cell types, including glioblastoma (GB), though no prior study has described the evaluation of CRISPR screening in GB in vivo. Here, we describe a protocol for targeting and transcriptionally repressing GB-specific long noncoding RNAs (lncRNAs) by CRISPR interference (CRISPRi) system in vivo, with tumor growth in the mouse cerebral cortex. Read More

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Generation of Drosophila attP containing cell lines using CRISPR-Cas9.

G3 (Bethesda) 2021 May 8. Epub 2021 May 8.

Drosophila Genomics Resource Center, Indiana University, Bloomington, Indiana 47405, USA.

The generation of Drosophila stable cell lines have become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Read More

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Lrp5 Mutant and Crispant Zebrafish Faithfully Model Human Osteoporosis, Establishing the Zebrafish as a Platform for CRISPR-Based Functional Screening of Osteoporosis Candidate Genes.

J Bone Miner Res 2021 May 6. Epub 2021 May 6.

Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Read More

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Minimized combinatorial CRISPR screens identify genetic interactions in autophagy.

Nucleic Acids Res 2021 Jun;49(10):5684-5704

Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.

Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. Read More

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Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.

Nature 2021 May 5. Epub 2021 May 5.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape. Read More

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Application of machine learning to large in vitro databases to identify drug-cancer cell interactions: azithromycin and KLK6 mutation status.

Oncogene 2021 May 5;40(21):3766-3770. Epub 2021 May 5.

Zephyr AI, Washington, DC, USA.

Recent advances in machine learning promise to yield novel insights by interrogation of large datasets ranging from gene expression and mutation data to CRISPR knockouts and drug screens. We combined existing and new algorithms with available experimental data to identify potentially clinically relevant relationships to provide a proof of principle for the promise of machine learning in oncological drug discovery. Specifically, we screened cell line data from the Cancer Dependency Map for the effects of azithromycin, which has been shown to kill cancer cells in vitro. Read More

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Comprehensive CRISPR-Cas9 screens identify genetic determinants of drug responsiveness in multiple myeloma.

Blood Adv 2021 05;5(9):2391-2402

Department of Internal Medicine III, Ulm University Hospital, Ulm, Germany.

The introduction of new drugs in the past years has substantially improved outcome in multiple myeloma (MM). However, the majority of patients eventually relapse and become resistant to one or multiple drugs. While the genetic landscape of relapsed/ resistant multiple myeloma has been elucidated, the causal relationship between relapse-specific gene mutations and the sensitivity to a given drug in MM has not systematically been evaluated. Read More

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CRISPR screen in cancer: status quo and future perspectives.

Am J Cancer Res 2021 15;11(4):1031-1050. Epub 2021 Apr 15.

Department of Medical Oncology, Jinling Hospital, The First School of Clinical Medicine, Southern Medical University Nanjing 210002, China.

Clustered regularly interspaced short palindromic repeats (CRISPR) system offers a powerful platform for genome manipulation, including protein-coding genes, noncoding RNAs and regulatory elements. The development of CRISPR screen enables high-throughput interrogation of gene functions in diverse tumor biologies, such as tumor growth, metastasis, synthetic lethal interactions, therapeutic resistance and immunotherapy response, which are mostly performed or in transplant models. Recently, direct CRISPR screens have been developed to identify drivers of tumorigenesis in native microenvironment. Read More

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Glycosylation Limits Forward Trafficking of the Tetraspan Membrane Protein PMP22.

J Biol Chem 2021 Apr 29:100719. Epub 2021 Apr 29.

Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37240, United States; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37240, United States. Electronic address:

Peripheral myelin protein 22 (PMP22) folds and trafficks inefficiently, with only 20% of newly expressed protein trafficking to the cell surface. This behavior is exacerbated in many of the mutants associated with Charcot-Marie-Tooth disease (CMTD), motivating further study. Here we characterized the role of N-glycosylation in limiting PMP22 trafficking. Read More

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SARS-CoV-2 RNA: Exclusive friends and common foes.

Cell 2021 04;184(9):2276-2278

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address:

Infection with SARS-CoV-2 sets off a molecular arms race between virus replication and host cell defense. In this issue of Cell, Flynn, Belk, et al. integrate an advanced large-scale RNA-centered approach with custom CRISPR screens to functionally characterize the interactome of the SARS-CoV-2 RNA genome during infection. Read More

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Profiling the genetic determinants of chromatin accessibility with scalable single-cell CRISPR screens.

Nat Biotechnol 2021 Apr 29. Epub 2021 Apr 29.

New York Genome Center, New York, NY, USA.

CRISPR screens have been used to connect genetic perturbations with changes in gene expression and phenotypes. Here we describe a CRISPR-based, single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC) to link genetic perturbations to genome-wide chromatin accessibility in a large number of cells. In human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 105 chromatin-related genes, generating chromatin accessibility data for ~30,000 single cells. Read More

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