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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
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Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening
1Department of Discovery Oncology, Genentech, South San Francisco, CA, United States of America.
Plos One
|December 29, 2016
Summary
Synthetic CRISPR reagents enable arrayed screening for DNA replication regulators. This method offers a viable alternative to pooled lentiviral screens, identifying significant hits efficiently.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- CRISPR-Cas9 screens are typically pooled, limiting applications.
- Arrayed lentiviral screening presents significant technical challenges.
Purpose of the Study:
- To evaluate synthetic CRISPR reagents for arrayed screening.
- To identify regulators of aberrant DNA replication using this approach.
Main Methods:
- Arrayed screening with synthetic CRISPR RNAs targeting GMNN and 640 ubiquitin-related genes.
- Assay for aberrant DNA replication in HCT-116 cells expressing Cas9.
- Validation using siRNA gene knockdown.
Main Results:
- Synthetic CRISPR RNAs induced statistically significant phenotypes for GMNN within 72 hours.
- Identified known and novel DNA replication regulators, with CRISPR outperforming siRNA screens.
- Demonstrated efficient GMNN protein loss and DNA editing.
Conclusions:
- Synthetic CRISPR reagents are effective for arrayed screening.
- This approach overcomes limitations of pooled lentiviral screens.
- Enables efficient identification of gene function in arrayed formats.
