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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
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Higher resolution pooled genome-wide CRISPR knockout screening in Drosophila cells using Integration and Anti-CRISPR
Raghuvir Viswanatha1, Samuel Entwisle1, Claire Hu1
1Department of Genetics, Harvard Medical School, Boston, MA 02115.
Biorxiv : the Preprint Server for Biology
|September 30, 2024
Summary
We developed IntAC, a new CRISPR screening method for Drosophila, enhancing accuracy by suppressing Cas9 activity during early sgRNA expression. This approach identifies essential genes with high precision, improving genotype-to-phenotype mapping.
Area of Science:
- Genetics
- Molecular Biology
- Biotechnology
Background:
- CRISPR screens are powerful tools for genotype-to-phenotype mapping.
- Previous Drosophila CRISPR screening methods faced accuracy limitations due to early Cas9 activity.
Purpose of the Study:
- To enhance the accuracy and comprehensiveness of CRISPR screens in Drosophila.
- To introduce a novel method, IntAC (integrase with anti-CRISPR), for improved CRISPR screening.
Main Methods:
- Co-transfection of plasmids expressing sgRNA libraries and the anti-CRISPR protein AcrIIa4.
- Utilizing the high-strength dU6:3 promoter for sgRNA expression.
- Implementing site-specific integration for sgRNA library introduction.
Main Results:
- The IntAC method significantly improved precision-recall of Drosophila fitness genes (90-95% retrieval within 5% error).
- Elevated sgRNA levels, enabled by IntAC, were key to improved screening accuracy.
- Demonstrated strong correlation between Drosophila and human fitness genes and highlighted paralog effects.
Conclusions:
- IntAC is a straightforward enhancement for Drosophila CRISPR screening, dramatically increasing accuracy.
- The method is potentially applicable to virus-free CRISPR screens in various cell types, including mammalian cells.
