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Genome-scale chemogenomic CRISPR screens in human cells using the TKOv3 library.

ABSTRACT: CRISPR-based genetic screens revolutionized our ability to genetically probe cell biology. We present a protocol to conduct genome-scale chemogenomic dropout CRISPR screens in the human RPE1-hTERT p53^-/- cell line. We use the TKOv3 library, which contains 70,948 sgRNAs targeting 18,053 genes. Here, we describe how to set up the screen, the reagents required, and how to sequence and analyze the results. This protocol can be customized for other libraries, cell lines, and sequencing instruments. For complete details on the use and execution of this protocol, please refer to Olivieri et al. (2020).

SUBMITTER: Olivieri M

PROVIDER: S-EPMC7868615 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Genome-scale chemogenomic CRISPR screens in human cells using the TKOv3 library.

Olivieri Michele M Durocher Daniel D

STAR protocols 20210205 1

CRISPR-based genetic screens revolutionized our ability to genetically probe cell biology. We present a protocol to conduct genome-scale chemogenomic dropout CRISPR screens in the human RPE1-hTERT p53<sup>-/-</sup> cell line. We use the TKOv3 library, which contains 70,948 sgRNAs targeting 18,053 genes. Here, we describe how to set up the screen, the reagents required, and how to sequence and analyze the results. This protocol can be customized for other libraries, cell lines, and sequencing ins ...[more]

PMID: 33598657

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Project description:The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

Dataset Information

Genome-scale chemogenomic CRISPR screens in human cells using the TKOv3 library.

Publications

Genome-scale chemogenomic CRISPR screens in human cells using the TKOv3 library.

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