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

Area of Science:

• Biotechnology
• Molecular Biology
• Genetics

Background:

• Genetically encoded single-cell barcodes are crucial for lineage tracing and genetic screens.
• Current methods like nucleic acid barcoding offer high diversity but are destructive and costly, while fluorescence barcoding is nondestructive but lacks diversity.

Purpose of the Study:

• To experimentally validate a theoretical model for generating high-diversity barcode libraries using fluorescent protein combinations.
• To create a nondestructive, fast, and inexpensive barcoding system for single-cell applications.

Main Methods:

• Generated a library of approximately 150 barcodes from two-way combinations of 18 fluorescent proteins.
• Utilized a pooled cloning strategy for library generation and validation.
• Employed spectral flow cytometry for experimental readout in single mammalian cells.

Main Results:

• Demonstrated excellent classification performance for most individual fluorescent proteins and evaluated barcodes, with true positive rates exceeding 99% for many.
• Identified mTFP1 as an exception in classification performance.
• Validated the library's compatibility with genetic screening and lineage tracing for hundreds of genes or clones.

Conclusions:

• This proof-of-concept study establishes a foundation for developing significantly larger fluorescent protein barcode libraries (potentially >10^5).
• The developed system offers a promising alternative for high-throughput genetic screening and lineage tracing with nondestructive, rapid readout.