@article {1233489,
	title = {Pooled CRISPR Screens in Drosophila Cells},
	journal = {Curr Protoc Mol Biol},
	volume = {129},
	year = {2019},
	month = {2019 Dec},
	pages = {e111},
	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. {\textcopyright} 2019 by John Wiley \&amp; 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.},
	issn = {1934-3647},
	doi = {10.1002/cpmb.111},
	author = {Viswanatha, Raghuvir and Brathwaite, Roderick and Hu, Yanhui and Li, Zhongchi and Rodiger, Jonathan and Merckaert, Pierre and Chung, Verena and Mohr, Stephanie E and Perrimon, Norbert}
}
