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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
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Protocol for performing pooled CRISPR-Cas9 loss-of-function screens
Alyssa J Mathiowetz1, Melissa A Roberts1, David W Morgens2
1Departments of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA.
STAR Protocols
|March 31, 2023
Summary
This protocol details pooled CRISPR-Cas9 loss-of-function screens for identifying gene modifiers. It covers library preparation, cell screening, and data analysis for rapid, affordable gene function evaluation.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Bioinformatics
Background:
- Pooled CRISPR-Cas9 libraries enable large-scale gene function analysis.
- Loss-of-function screens are crucial for identifying genetic modifiers.
- Previous methods may lack speed, affordability, or global reach.
Purpose of the Study:
- To present a detailed protocol for pooled CRISPR-Cas9 loss-of-function screens.
- To enable identification of genetic modifiers using phenotypic readouts.
- To provide a comprehensive guide for library design, cell screening, and data analysis.
Main Methods:
- Design and amplification of pooled CRISPR-Cas9 libraries.
- Generation and screening of cells with phenotypic readouts (fluorescence or cell death).
- Deep sequencing and statistical analysis using cas9 High Throughput maximum Likelihood Estimator (HTMLE).
Main Results:
- The protocol facilitates rapid and affordable evaluation of gene functions globally.
- It enables the identification of genetic modifiers through specific phenotypic readouts.
- Detailed steps for library preparation, cell screening, and data analysis are provided.
Conclusions:
- This protocol offers a robust method for performing pooled CRISPR-Cas9 loss-of-function screens.
- It is applicable for identifying genetic modifiers across various biological contexts.
- The method enhances the efficiency and accessibility of large-scale genetic screens.
