Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Pooled loss- and gain-of-function CRISPR screening is widely used for mammalian gene function studies.
• Existing methods often overlook cellular heterogeneity, analyzing bulk cell populations instead of individual clonal responses.

Purpose of the Study:

• To develop and validate a novel barcoding approach to account for cellular heterogeneity in CRISPR screens.
• To investigate the impact of clonal cell line variations on pooled genetic screen outcomes.

Main Methods:

• Development of multi-level barcoded sgRNA libraries for tracing multiple clonal Cas9 cell lines.
• Application of barcoding to monitor growth kinetics and treatment responses under identical conditions.
• Enabling in-sample replication and sub-clonal lineage tracing within cells expressing the same sgRNA.

Main Results:

• The developed method allows for the simultaneous monitoring of multiplexed clonal cell lines.
• It enables precise tracking of growth kinetics and treatment responses across different clonal lines.
• Demonstrated how clonal heterogeneity in growth and response can significantly affect pooled screen analyses.

Conclusions:

• Cellular heterogeneity, specifically variations in growth kinetics and treatment response among clonal cell lines, can impair the accuracy of pooled genetic screens.
• The multi-level barcoding approach provides a robust method to identify and mitigate the effects of such heterogeneity.
• This methodology enhances the reliability of CRISPR screening for systematic gene function investigation.