Pooled CRISPR screening of high-content cellular phenotypes using ghost cytometry.

Area of Science:

• Cell Biology
• Genomics
• Biotechnology

Background:

• Image-based pooled CRISPR screening advances genotype-phenotype mapping in mammalian cells.
• Rapid cell enrichment based on morphology is a challenge for high-content screening.
• Existing methods limit large-scale gene perturbation screening for diverse cellular phenotypes.

Purpose of the Study:

• To demonstrate multimodal ghost cytometry for rapid pooled CRISPR screening.
• To apply fluorescent and label-free high-content phenotypes for cell sorting.
• To overcome limitations in screening vast cell populations for specific phenotypes.

Main Methods:

• Utilized multimodal ghost cytometry with fluorescent and label-free modes.
• Performed kinase-specific CRISPR screening targeting RelA nuclear translocation using fluorescence.
• Conducted large-scale screening for macrophage polarization genes using label-free mode.

Main Results:

• Successfully executed kinase-specific CRISPR screening for RelA translocation.
• Identified genes involved in macrophage polarization via label-free screening.
• Demonstrated label-free enrichment without invasive staining, preserving cells for downstream assays.

Conclusions:

• Multimodal ghost cytometry enables rapid pooled CRISPR screening across diverse phenotypes.
• Label-free ghost cytometry expands screening potential, especially when markers are absent.
• This method preserves cells, facilitating further downstream biological assays.