Functionalized Lineage Tracing for the Study and Manipulation of Heterogeneous Cell Populations.

Area of Science:

• Cell Biology
• Genetics
• Molecular Biology

Background:

• Understanding cellular processes in heterogeneous populations requires advanced lineage tracking beyond single-cell RNA sequencing.
• Current methods lack the ability to precisely identify and isolate specific cell lineages for in-depth study.

Purpose of the Study:

• To introduce a novel method, Control of Lineages by Barcode Enabled Recombinant Transcription (COLBERT), for tracking and isolating specific cell lineages.
• To enable high-resolution analysis of cellular processes in both normal and pathogenic states.

Main Methods:

• COLBERT utilizes unique single guide RNA (sgRNA) barcodes as stable, actively transcribed functional tags within cell lineages.
• These sgRNA barcodes are integrated into the genome, ensuring all progeny inherit the parental barcode.
• Lineage isolation is achieved using CRISPR activation (e.g., dCas9-VPR) with a barcode-matched sequence to trigger a fluorescent reporter.

Main Results:

• The COLBERT method allows for the stable integration and transcription of sgRNA barcodes in cellular progeny.
• Precise identification and isolation of specific lineages are achieved by matching the sgRNA barcode to a CRISPR activation system.
• This enables the selective enrichment of desired cell lineages from complex, heterogeneous populations.

Conclusions:

• COLBERT provides a powerful tool for high-resolution lineage tracing and isolation.
• The method enhances the ability to study cellular dynamics and understand disease mechanisms.
• COLBERT offers a significant advancement over existing single-cell analysis techniques.