Abstract
The discovery and implementation of CRISPR/Cas9 tools in pooled genetic screens have allowed for the rapid, high-fidelity identification of host-virus interactions. However, pooled CRISPR screening has significant limitations in its ability both to perform cell biology and plate reader-based screens and to find alleles that result in intermediate-strength phenotypes. Here we introduce an arrayed CRISPR screening method, FACS-IT, which allows researchers to use high content imaging analysis, plate reader assays, cell supernatant characterization, and percent infectivity to characterize CRISPR-mediated gene disruptions causing both moderate and extreme phenotypic changes. By using flow sorting capabilities and CRISPR libraries that are widely available, FACS-IT overcomes both the significant limitation of pooled screening approaches and the prohibitive costs of large-scale arrayed CRISPR reagents. In doing so, FACS-IT will enable researchers to creatively use CRISPR screening to obtain a deeper understanding of biology across a wide range of fields and applications.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Perreira JM, Meraner P, Brass AL (2016) Functional genomic strategies for elucidating human-virus interactions: will CRISPR knockout RNAi and haploid cells? Adv Virus Res 94:1–51
McDougall WM et al (2018) CRISPR genetic screens to discover host-virus interactions. Curr Opin Virol 29:87–100
Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096
Jinek M et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821
Perez-Pinera P et al (2013) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 10(10):973–976
Gilbert LA et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451
Sanson KR et al (2018) Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9(1):5416
Gilbert LA et al (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159(3):647–661
Horlbeck MA et al (2016) Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Elife 5. https://doi.org/10.7554/eLife.19760
Konermann S et al (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517(7536):583–588
Savidis G et al (2016) Identification of Zika virus and dengue virus dependency factors using functional genomics. Cell Rep 16(1):232–246
Ma H et al (2015) A CRISPR-based screen identifies genes essential for West-Nile-virus-induced cell death. Cell Rep 12(4):673–683
Marceau CD et al (2016) Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens. Nature 535(7610):159–163
Acknowledgments
This work was supported by an Investigators in the Pathogenesis of Infectious Disease grant from the Burroughs Wellcome Fund to A.L.B.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
McDougall, W.M., Kandpal, M., Perreira, J.M., Brass, A.L. (2020). Discovery of Zika Virus Dependency and Restriction Factors Using Flow-Based Arrayed CRISPR Screening for Identification of Targets (FACS-IT). In: Kobinger, G., Racine, T. (eds) Zika Virus. Methods in Molecular Biology, vol 2142. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0581-3_17
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0581-3_17
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0580-6
Online ISBN: 978-1-0716-0581-3
eBook Packages: Springer Protocols