Pooled CRISPR Screens in Drosophila Cells.
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 â¼3 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.
Palavras-chave
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
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Sistemas CRISPR-Cas
Tipo de estudo:
Observational_studies
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article