Transfection, pool maintenance, and drug addition

Link to detailed protocol: https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cpmb.111

Pooled CRISPR Screens in Drosophila Cells

Raghuvir Viswanatha 

Roderick Brathwaite 

Yanhui Hu 

Zhongchi Li 

Jonathan Rodiger 

Pierre Merckaert 

Verena Chung 

Stephanie E. Mohr 

Norbert Perrimon

First published: 06 November 2019

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. © 2019 by John Wiley & Sons, Inc.

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