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High-content imaging-based pooled CRISPR screens in mammalian cells
Name
jcb_202008158.pdf
Description
Published version
Size
5.93 MB
Format
Adobe PDF
Checksum (MD5)
8fcb232d66ee9a3be13764d0b8de9a8e
Author(s)
Yan, Xiaowei
Stuurman, Nico
Ribeiro, Susana A
Tanenbaum, Marvin E
Horlbeck, Max A
Liem, Christina R
Jost, Marco
Weissman, Jonathan S
Vale, Ronald D
Date Issued
2021
Journal
The Journal of Cell Biology
Publisher
Rockefeller University Press
Version
Final published version
Abstract
CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.
Terms of Use
Creative Commons Attribution 4.0 International license
Persistent DSpace Link
DOI of Published Version
10.1083/JCB.202008158
