272 results match your criteria pooled crispr

In vivo CRISPR-Cas9 knockout screening using quantitative PCR identifies thymosin beta-4 X-linked that promotes diffuse-type gastric cancer metastasis.

Mol Carcinog 2021 Jun 3. Epub 2021 Jun 3.

Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, ChunCheon-si, Gangwon-do, South Korea.

Gastric cancer (GC) is histologically classified into intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), and the latter is poorly differentiated and highly metastatic. In this study, using quantitative real-time polymerase chain reaction, we described a complete protocol for in vivo CRISPR-Cas9-based knockout screening of essential genes for DGC metastasis. We functionally screened 30 candidate genes using our mouse DGC models lacking Smad4, p53, and E-cadherin. Read More

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High-throughput CRISPR-mediated 3D enrichment platform for functional interrogation of chemotherapeutic resistance.

Biotechnol Bioeng 2021 May 29. Epub 2021 May 29.

Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.

Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. 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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Population genomics of invasive rodents on islands: Genetic consequences of colonization and prospects for localized synthetic gene drive.

Evol Appl 2021 May 10;14(5):1421-1435. Epub 2021 Mar 10.

National Wildlife Research Center USDA APHIS Wildlife Services Fort Collins Colorado USA.

Introduced rodent populations pose significant threats worldwide, with particularly severe impacts on islands. Advancements in genome editing have motivated interest in synthetic gene drives that could potentially provide efficient and localized suppression of invasive rodent populations. Application of such technologies will require rigorous population genomic surveys to evaluate population connectivity, taxonomic identification, and to inform design of gene drive localization mechanisms. 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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Pooled CRISPR screening identifies mA as a positive regulator of macrophage activation.

Sci Adv 2021 Apr 28;7(18). Epub 2021 Apr 28.

Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

mA RNA modification is implicated in multiple cellular responses. However, its function in the innate immune cells is poorly understood. Here, we identified major mA "writers" as the top candidate genes regulating macrophage activation by LPS in an RNA binding protein focused CRISPR screening. Read More

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High-throughput functional variant screens via in vivo production of single-stranded DNA.

Proc Natl Acad Sci U S A 2021 May;118(18)

Department of Genetics, Harvard Medical School, Boston, MA 02115.

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. Read More

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Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells.

STAR Protoc 2021 Jun 9;2(2):100426. Epub 2021 Apr 9.

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK.

CRISPR/Cas9 screens are a powerful approach to identify key regulators of biological processes. By combining pooled CRISPR/Cas9 screening with single-cell RNA-sequencing readout, individual perturbations can be assessed in parallel both comprehensively and at scale. Importantly, this allows gene function and regulation to be interrogated at a cellular level in an unbiased manner. Read More

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GOANA: A Universal High-Throughput Web Service for Assessing and Comparing the Outcome and Efficiency of Genome Editing Experiments.

CRISPR J 2021 Apr;4(2):243-252

Australian e-Health Research Centre, Commonwealth Scientific and Industrial Research Organization, North Ryde, Australia; Department of Biomedical Sciences, Macquarie Park, Australia.

The increased development of functionally diverse and highly specialized genome editors has created the need for comparative analytics tools that are able to profile the mutational outcomes, particularly rare and complex outcomes, to assess the editor's applicability to different domains. To address this need, we have developed Generalizable On-target activity ANAlyzer (GOANA), a high-throughput web-based software for determining editing efficiency and cataloguing rare outcomes from next-generation sequencing data. GOANA calculates mutation frequency and outcomes relative to a supplied control sample. Read More

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NSDHL promotes triple-negative breast cancer metastasis through the TGFβ signaling pathway and cholesterol biosynthesis.

Breast Cancer Res Treat 2021 Jun 16;187(2):349-362. Epub 2021 Apr 16.

Key Laboratory of Breast Cancer in Shanghai, Department of Breast Surgery, Fudan University Shanghai Cancer Center, 270 Dong-An Road, Build 7, Room 303, Shanghai, 200032, China.

Purpose: Metastasis is the main cause of breast cancer mortality. Recent studies have proved that lipid metabolic reprogramming plays critical roles in breast cancer carcinogenesis and metastasis. We aim to identify critical lipid metabolism genes in breast cancer metastasis. Read More

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A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

Metab Eng 2021 Jul 1;66:114-122. Epub 2021 Apr 1.

The Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, USA; Department of Pediatrics, University of California, San Diego, USA; Department of Bioengineering, University of California, San Diego, USA; National Biologics Facility, Technical University of Denmark, Denmark. Electronic address:

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Read More

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A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast.

BMC Genomics 2021 Mar 23;22(1):205. Epub 2021 Mar 23.

Department of Molecular and Cell Biology, Berkeley, CA, 94720, USA.

Background: CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis.

Results: We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Read More

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Androgen Receptor Regulates CD44 Expression in Bladder Cancer.

Cancer Res 2021 Jun 9;81(11):2833-2846. Epub 2021 Mar 9.

Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California.

The androgen receptor (AR) is important in the development of both experimental and human bladder cancer. However, the role of AR in bladder cancer growth and progression is less clear, with literature indicating that more advanced stage and grade disease are associated with reduced AR expression. To determine the mechanisms underlying these relationships, we profiled AR-expressing human bladder cancer cells by AR chromatin immunoprecipitation sequencing and complementary transcriptomic approaches in response to stimulation by the synthetic androgen R1881. Read More

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HIV-CRISPR: A CRISPR/Cas9 Screening Method to Identify Genes Affecting HIV Replication.

Bio Protoc 2020 May 5;10(9):e3614. Epub 2020 May 5.

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

Screening with CRISPR/Cas9 technology has already led to significant discoveries in the fields of cancer biology, cell biology and virology. Because of the relatively low false discovery rates and the ability to perform high-throughput, pooled approaches, it has rapidly become the assay of choice for screening studies, including whole-genome screens. Here, we describe a CRISPR screening protocol that allows for efficient screening of the entire life cycle of HIV-1 through packaging of the HIV-CRISPR lentiviral genomes by infecting HIV-1 virus . Read More

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Characterizing the molecular regulation of inhibitory immune checkpoints with multimodal single-cell screens.

Nat Genet 2021 03 1;53(3):322-331. Epub 2021 Mar 1.

Center for Genomics and Systems Biology, New York University, New York, NY, USA.

The expression of inhibitory immune checkpoint molecules, such as programmed death-ligand (PD-L)1, is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here, we apply expanded CRISPR-compatible (EC)CITE-seq, a technology that combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Read More

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Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion.

Nat Genet 2021 03 1;53(3):332-341. Epub 2021 Mar 1.

Columbia Center for Translational Immunology, New York, NY, USA.

Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). Read More

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In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8 T cell fate decisions.

Cell 2021 Mar 25;184(5):1245-1261.e21. Epub 2021 Feb 25.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address:

How early events in effector T cell (T) subsets tune memory T cell (T) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of T and T cells using in vivo pooled CRISPR screening, focusing on negative regulators of T responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of T differentiation, in part through modulating mTORC1 signaling. Read More

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Accelerating target deconvolution for therapeutic antibody candidates using highly parallelized genome editing.

Nat Commun 2021 02 24;12(1):1277. Epub 2021 Feb 24.

Department of Laboratory Medicine, Hematology and Transfusion Medicine, Lund, Sweden.

Therapeutic antibodies are transforming the treatment of cancer and autoimmune diseases. Today, a key challenge is finding antibodies against new targets. Phenotypic discovery promises to achieve this by enabling discovery of antibodies with therapeutic potential without specifying the molecular target a priori. Read More

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February 2021

Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival.

Nat Commun 2021 02 23;12(1):1244. Epub 2021 Feb 23.

York Biomedical Research Institute, University of York, York, UK.

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Read More

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February 2021

Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages.

Sci Rep 2021 Feb 19;11(1):4245. Epub 2021 Feb 19.

Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, UK.

Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Read More

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February 2021

Massively parallel assessment of human variants with base editor screens.

Cell 2021 Feb;184(4):1064-1080.e20

Genetic Perturbation Platform, Broad Institute, Cambridge, MA 02142, USA. Electronic address:

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. Read More

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February 2021

CRISPR base editor screens identify variant function at scale.

Mol Cell 2021 02;81(4):647-648

Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Computational Biology Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Genome Sciences, University of Washington, Seattle, WA, USA. Electronic address:

Cuella-Martin et al. (2021) and Hanna et al. (2021) showcase CRISPR base editing in large-scale pooled screens in human cells to discover both loss- and gain-of-function variants, enabling protein structure/function insights and clinical variant interpretation. Read More

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February 2021

Advanced single-cell pooled CRISPR screening identifies C19orf53 required for cell proliferation based on mTORC1 regulators.

Cell Biol Toxicol 2021 Feb 13. Epub 2021 Feb 13.

College of Animal Science and Technology, Hunan Agricultural University, Changsha, 410128, China.

Multiplexed single-cell CRISPR screening has accelerated the systematic dissection of biological discoveries; however, the efficiency of CRISPR-based gene knockout has inherent limitations. Here, we present DoNick-seq, an advanced method for facilitating gene knockout and reducing off-target activity. We re-engineered two popular plasmid constructs suitable for use in pooled CRISPR screening of the single-cell transcriptome. Read More

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February 2021

Signal peptidase complex subunit 1 is an essential Zika virus host factor in placental trophoblasts.

Virus Res 2021 Apr 9;296:198338. Epub 2021 Feb 9.

Institute of Experimental Internal Medicine, Otto Von Guericke University Magdeburg, 39120, Magdeburg, Germany.

Zika is a major teratogenic virus that can be transmitted from pregnant women to the fetus via the transplacental route. At present, no specific vaccines or treatments are available. Large-scale functional genomics approaches for the analysis of host cell function in infection greatly improve the understanding of molecular infection processes and advance the discovery of antiviral targets. Read More

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Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing.

NAR Genom Bioinform 2020 Jun 13;2(2):lqaa034. Epub 2020 May 13.

Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.

The libraries generated by high-throughput single cell RNA-sequencing (scRNA-seq) platforms such as the Chromium from 10× Genomics require considerable amounts of sequencing, typically due to the large number of cells. The ability to use these data to address biological questions is directly impacted by the quality of the sequence data. Here we have compared the performance of the Illumina NextSeq 500 and NovaSeq 6000 against the BGI MGISEQ-2000 platform using identical Single Cell 3' libraries consisting of over 70 000 cells generated on the 10× Genomics Chromium platform. Read More

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Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs.

Genome Res 2021 Mar 11;31(3):461-471. Epub 2021 Feb 11.

Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. Read More

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Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton.

Plant Methods 2021 Feb 8;17(1):16. Epub 2021 Feb 8.

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.

Background: Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. Read More

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February 2021

A Generally Applicable CRISPR/Cas9 Screening Technique to Identify Host Genes Required for Virus Infection as Applied to Human Cytomegalovirus (HCMV) Infection of Epithelial Cells.

Methods Mol Biol 2021 ;2244:247-264

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens enable virus-host genetic screens to be undertaken in a more robust manner than previously possible and has had a tremendous impact in the field of virus study. Researchers can take advantage of the power of CRISPR genetic screens to discover virus-host interaction genes including host receptors and signaling molecules (Bazzone et al., mBio 10 (1): e02734-18, 2019; E et al. Read More

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Targeting oncoproteins with a positive selection assay for protein degraders.

Sci Adv 2021 Feb 5;7(6). Epub 2021 Feb 5.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Read More

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February 2021