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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Katherine Chan1, Amy Hin Yan Tong1, Kevin R Brown1
1Donnelly Centre, University of Toronto.
Journal of Visualized Experiments : Jove
|September 24, 2019
Summary
This study details a protocol for pooled genome-wide CRISPR-Cas9 screens in human cells. These screens enable unbiased identification of genes essential for cellular functions and potential cancer therapies.
Area of Science:
- Genomics
- Molecular Biology
- Functional Genomics
Background:
- CRISPR-Cas9 technology enables precise genome editing.
- Genome-wide genetic screens are crucial for functional genomics in mammalian cells.
- CRISPR-Cas screens offer an efficient and cost-effective method for phenotype analysis.
Purpose of the Study:
- To provide a detailed protocol for performing pooled genome-wide CRISPR-Cas9 screens in human cells.
- To facilitate unbiased, systematic identification of gene functions and cellular processes.
- To uncover contextually essential genes in tumor cells for targeted cancer therapies.
Main Methods:
- Utilizing libraries of single-guide RNAs (sgRNAs) targeting open reading frames.
- Generating thousands of genetic perturbations in a pooled cell population.
- Performing differential analysis of screening data across various cell lines and cancer types.
Main Results:
- CRISPR-Cas screens successfully uncover genetic blueprints for cellular phenotypes.
- Differential analysis identifies contextually essential genes in tumor cells.
- The protocol addresses practical challenges in large-scale human cell screening.
Conclusions:
- Pooled genome-wide CRISPR-Cas9 screens are a powerful tool for functional genomics.
- This protocol simplifies the complex process of large-scale genetic screens in human cells.
- The findings have implications for discovering novel anticancer therapeutic targets.
