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Pooled CRISPR Screens in Drosophila Cells.
Viswanatha, Raghuvir; Brathwaite, Roderick; Hu, Yanhui; Li, Zhongchi; Rodiger, Jonathan; Merckaert, Pierre; Chung, Verena; Mohr, Stephanie E; Perrimon, Norbert.
Afiliação
  • Viswanatha R; Department of Genetics, Harvard Medical School, Boston, Massachusetts.
  • Brathwaite R; Department of Genetics, Harvard Medical School, Boston, Massachusetts.
  • Hu Y; Drosophila RNAi Screening Center, Harvard Medical School, Boston, Massachusetts.
  • Li Z; Department of Genetics, Harvard Medical School, Boston, Massachusetts.
  • Rodiger J; Drosophila RNAi Screening Center, Harvard Medical School, Boston, Massachusetts.
  • Merckaert P; Department of Genetics, Harvard Medical School, Boston, Massachusetts.
  • Chung V; Department of Genetics, Harvard Medical School, Boston, Massachusetts.
  • Mohr SE; Drosophila RNAi Screening Center, Harvard Medical School, Boston, Massachusetts.
  • Perrimon N; Department of Genetics, Harvard Medical School, Boston, Massachusetts.
Curr Protoc Mol Biol ; 129(1): e111, 2019 12.
Article em En | MEDLINE | ID: mdl-31763777
High-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow-up studies performed in vivo in Drosophila, mammalian cells, and/or other systems. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Pooled-format screening with Cas9-expressing Drosophila S2R+ cells in the presence of cytotoxin Support Protocol 1: Optimization of cytotoxin concentration for Drosophila cell screening Support Protocol 2: CRISPR sgRNA library design and production for Drosophila cell screening Support Protocol 3: Barcode deconvolution and analysis of screening data.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: RNA Guia de Cinetoplastídeos / Ensaios de Triagem em Larga Escala / Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas / Sistemas CRISPR-Cas Tipo de estudo: Observational_studies / Prognostic_studies Limite: Animals Idioma: En Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: RNA Guia de Cinetoplastídeos / Ensaios de Triagem em Larga Escala / Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas / Sistemas CRISPR-Cas Tipo de estudo: Observational_studies / Prognostic_studies Limite: Animals Idioma: En Ano de publicação: 2019 Tipo de documento: Article