Optimization of Genome-Wide CRISPR Screens Using Dual-Guide RNA Infection with Cas9 Electroporation (DICE)

Area of Science:

• Molecular Biology
• Genetics
• Immunology

Background:

• CRISPR screening is vital for functional genomics.
• Existing methods like SLICE have limitations in scalability and robustness.
• Primary cells often require transient Cas9 expression for CRISPR applications.

Purpose of the Study:

• To introduce and validate a novel CRISPR screening method, DICE.
• To overcome the limitations of SLICE in scalability and robustness.
• To assess DICE's efficacy in identifying essential genes and PD-L1 modulators.

Main Methods:

• Development of dual guide RNA lentiviral constructs for co-expression.
• Integration of Cas9 protein electroporation with lentiviral delivery.
• Application of DICE in genome-wide screens using Interferon-gamma-activated THP1 cells.

Main Results:

• DICE demonstrated superior performance compared to SLICE in genome-wide screens.
• The method successfully identified essential genes.
• DICE effectively pinpointed modulators of PD-L1 expression.

Conclusions:

• DICE offers a scalable and robust alternative for CRISPR screens in primary cells.
• The technique is suitable for cell types requiring transient Cas9 expression.
• DICE maintains screening quality while enabling reduced-scale experiments.