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Multiplexed Perturbation Enables Scalable Pooled Screens

ABSTRACT: CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale. However, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of multiplexed sgRNA delivery via high multiplicity of infection (MOI) in pooled CRISPR interference (CRISPRi) screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation, and collision rates. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of intracellular adhesion molecule ICAM-1, successfully identifying novel candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.

ORGANISM(S): Homo sapiens

PROVIDER: GSE302335 | GEO | 2026/01/16

REPOSITORIES: GEO

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