Genetically-Encoded Fluorescence Barcodes Allow for Single-Cell Analysis via Spectral Flow Cytometry.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Genetically-encoded single-cell barcodes are crucial for lineage tracing and genetic screens.
• Existing nucleic acid methods offer high diversity but have slow, destructive readouts.
• Current fluorescence methods are non-destructive and fast but lack sufficient diversity.

Purpose of the Study:

• To experimentally validate a theoretical framework for high-diversity fluorescent protein barcode libraries.
• To develop a non-destructive, fast, and inexpensive barcoding system for biological applications.
• To establish a foundation for creating significantly larger barcode libraries.

Main Methods:

• Generated a library of barcodes using two-way combinations of 18 fluorescent proteins.
• Employed a pooled cloning strategy for library construction and validation.
• Utilized spectral flow cytometry for experimental readout in single mammalian cells.

Main Results:

• Successfully created and validated a library of approximately 150 barcodes (61 tested experimentally).
• Demonstrated excellent classification performance for most fluorescent proteins and barcodes (>99% true positive rate).
• Identified mTFP1 as an exception with lower classification performance.

Conclusions:

• Fluorescent protein combinations can generate high-diversity barcode libraries with non-destructive, rapid readout.
• This proof-of-concept supports applications in genetic screening and lineage tracing for hundreds of genes/clones.
• The approach provides a scalable foundation for libraries with potential diversity exceeding 10^5.