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Transfection, pool maintenance, and drug addition
Last updated date: Dec 7, 2020 Views: 968 Forks: 0
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Link to detailed protocol: https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cpmb.111
Pooled CRISPR Screens in Drosophila Cells
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.
- Viswanatha, R and Perrimon, N(2020). Transfection, pool maintenance, and drug addition. Bio-protocol Preprint. bio-protocol.org/prep685.
- Viswanatha, R., Li, Z., Hu, Y. and Perrimon, N.(2018). Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells. eLife. DOI: 10.7554/eLife.36333
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