Multiplexed Perturbation Enables Scalable Pooled Screens

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. Overall design: K-562 cells where infected with CRISPRi guide RNA libraries at varying multiplicity of infection (MOI) to test the effect of guide RNA multiplexing on CRISPR screen performance. After transduction, cells were grown for 14 days with or without selection by imatinib treatment prior to sequencing the guide RNA abundances. In a second experiment, transduced cells were stained for CD54 (ICAM-1) and sorted into high, low and medium CD54-expressing cell prior to sequencing the guide RNA abundances.