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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
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High-content imaging-based pooled CRISPR screens in mammalian cells.
Xiaowei Yan1, Nico Stuurman1, Susana A Ribeiro1,2
1Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA.
The Journal of Cell Biology
|January 19, 2021
Summary
This study introduces optical enrichment, a microscopy-based method for pooled CRISPR screens. It enables automated identification and isolation of cells with specific CRISPR-induced phenotypes, advancing genetic screening capabilities.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- CRISPR gene inactivation links genes to cellular phenotypes.
- Pooled CRISPR screens using sgRNAs are limited to selectable phenotypes.
- A need exists for screening phenotypes not amenable to traditional selection methods.
Purpose of the Study:
- To develop a microscopy-based approach for pooled CRISPR screens.
- To enable the selection of cells based on visually identifiable CRISPR-induced phenotypes.
- To expand the scope of pooled CRISPR screening beyond chemical or FACS enrichment.
Main Methods:
- Developed 'optical enrichment,' a microscopy-based screening method.
- Automated image analysis identifies cells with specific CRISPR phenotypes.
- Photoactivation marks selected cells for isolation via fluorescence-activated cell sorting (FACS).
- A μManager plugin facilitates automated cell identification and photoactivation.
Main Results:
- Screened ~1.5 million cells in 8 hours.
- Successfully screened 6,092 sgRNAs targeting 544 genes for nuclear size regulation.
- Identified 14 bona fide genes affecting nuclear size.
Conclusions:
- Optical enrichment provides a scalable method for imaging-based pooled CRISPR screens.
- This approach significantly expands the types of phenotypes that can be screened using CRISPR.
- Facilitates high-throughput genetic screens for visually defined cellular characteristics.
