Genetic screens in human cells using the CRISPR-Cas9 system

Area of Science:

• Genetics
• Molecular Biology
• Genome Editing

Background:

• The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has revolutionized mammalian genetics.
• Existing tools allow for rapid generation of genetically modified cell lines and animals.

Purpose of the Study:

• To develop and validate a pooled, loss-of-function genetic screening approach using a genome-scale lentiviral single-guide RNA (sgRNA) library.
• To assess the utility of this screening method for both positive and negative selection in mammalian cells.

Main Methods:

• A genome-scale lentiviral sgRNA library with 73,000 sgRNAs was used to generate knockout collections in two human cell lines.
• Pooled genetic screens were performed for resistance to 6-thioguanine and etoposide, and for essential genes via negative selection.
• Massively parallel sequencing was employed to track the complex mutant pool after stable integration of sgRNA expression cassettes.

Main Results:

• A screen for 6-thioguanine resistance successfully identified components of the DNA mismatch repair pathway.
• A screen for etoposide resistance identified TOP2A and CDK6.
• Negative selection screens identified gene sets involved in fundamental cellular processes, and sgRNA efficiency was linked to specific sequence motifs.

Conclusions:

• Cas9/sgRNA screens represent a powerful and systematic approach for genetic analysis in mammalian cells.
• The developed screening method is effective for both positive and negative selection screens.
• Predicting effective sgRNAs based on sequence motifs can enhance screening efficiency.