| Literature DB >> 31763777 |
Raghuvir Viswanatha1, Roderick Brathwaite1,2, Yanhui Hu1,2, Zhongchi Li1, Jonathan Rodiger1,2, Pierre Merckaert1,2, Verena Chung1,2, Stephanie E Mohr1,2, Norbert Perrimon1,2,3.
Abstract
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.Entities:
Keywords: CRISPR; Drosophila; functional genomics; high-throughput screening; pooled-format screening
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Year: 2019 PMID： 31763777 PMCID： PMC6961806 DOI： 10.1002/cpmb.111
Source DB: PubMed Journal: Curr Protoc Mol Biol ISSN： 1934-3647