Scalable imaging-based profiling of CRISPR perturbations with protein barcodes

Area of Science:

• Genomics
• Cell Biology
• Bioinformatics

Background:

• Imaging-based CRISPR screens are powerful for functional genomics.
• Protein barcodes offer cost-effective, imaging-compatible barcoding for pooled perturbations.
• Existing methods face scalability challenges due to complex cloning and decoding.

Purpose of the Study:

• To introduce poolVis and cellPool, an integrated experimental and computational platform.
• To overcome limitations in scalability and confidence for protein barcode-based CRISPR screens.
• To enable high-content functional genomics with improved efficiency and accuracy.

Main Methods:

• poolVis utilizes Cre-lox-mediated reconfiguration for efficient barcode-sgRNA pairing during viral integration.
• cellPool employs a scalable computational workflow for decoding protein barcodes from large image datasets.
• The platform was applied to CRISPR interference (CRISPRi) profiling in MCF10A cells.

Main Results:

• Reduced barcode shuffling during pooled cloning and delivery.
• Generation of unpooled image galleries from multi-terabyte datasets.
• Uncovered known and novel phenotypes, including nuclear morphology changes and DNA damage epistasis.

Conclusions:

• The poolVis and cellPool platform significantly enhances the scalability and reliability of protein barcode-based CRISPR screens.
• This integrated approach facilitates high-content functional genomics and the discovery of complex cellular phenotypes.
• The findings advance the study of gene function and cellular responses to genetic perturbations.