Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen

Area of Science:

• Vascular Biology
• Cell Biology
• Genomics

Background:

• Endothelial cell monolayer integrity is vital for vascular and lymphatic system function.
• Existing methods for identifying junction-regulating genes are not compatible with high-throughput screening.

Purpose of the Study:

• To develop a novel, high-throughput assay for identifying genes that regulate endothelial cell permeability.
• To overcome limitations of current assays for genome-wide genetic screens.

Main Methods:

• Developed a microcarrier-based assay (MAPs) where endothelial cells form monolayers on microcarriers.
• Validated MAP sensitivity against thrombin-induced permeability changes, comparable to impedance measurements.
• Utilized fluorescence-assisted sorting to distinguish cells based on gene expression (sgRNAs).

Main Results:

• Minimized false-positive results in the MAP assay.
• Demonstrated effective blockade of thrombin-induced junction disassembly using sgRNAs targeting known signaling proteins.
• Successfully separated MAPs carrying specific sgRNAs via fluorescence-assisted sorting.

Conclusions:

• MAPs are suitable for high-throughput experimentation and genome-wide screens.
• The assay can identify genes mediating thrombin's disruptive effects on endothelial cell junctions.
• This technology advances the study of vascular and lymphatic system permeability regulation.