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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
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Increasing the performance of pooled CRISPR-Cas9 drop-out screening
Benedict C S Cross1, Steffen Lawo1, Caroline R Archer1
1Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom.
Scientific Reports
|August 23, 2016
Summary
A simple CRISPR-Cas tracrRNA modification enhances genetic screening robustness. This improves guide RNA stability and hit scoring, creating a powerful new platform for drug target discovery.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas systems are powerful tools for genetic engineering in eukaryotic cells.
- Applications include cell line engineering, animal model generation, and functional genomic screens.
- Pooled genetic screening with CRISPR-Cas offers new avenues for drug target identification and validation.
Purpose of the Study:
- To assess CRISPR-Cas screening functionality across different cell lines.
- To identify improvements that enhance screening sensitivity.
- To optimize CRISPR-Cas screening for drug target discovery.
Main Methods:
- Construction of ultra-complex libraries with sgRNA sequences targeting essential genes.
- Evaluation of screening performance in haploid and hypotriploid cell lines.
- Comparison of two guide design algorithms and two tracrRNA variants in time-resolved analysis.
Main Results:
- A simple adaptation of the tracrRNA significantly improved guide RNA retention during screening.
- This modification reduced the need for multiple sgRNAs per gene.
- Enhanced robustness led to increased hit scoring and improved screening outcomes.
Conclusions:
- A modified tracrRNA is a key adaptation for robust CRISPR-Cas screening.
- This improvement enhances the power of CRISPR-Cas for functional genomic screens.
- The optimized platform facilitates efficient drug target identification and validation.
