Lu Xiaoming, Pritko Daniel J, Abravanel Megan E, Huggins Jonah R, Ogunleye Oluwaferanmi, Biswas Tirthankar, Ashy Katia C, Woods Semaj K, Livingston Mariclaire W T, Blenner Mark A, Birtwistle Marc R
Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina 29634, United States.
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware 19716, United States.
ACS Synth Biol. 2025 May 16;14(5):1533-1548. doi: 10.1021/acssynbio.4c00807. Epub 2025 May 6.
Genetically encoded, single-cell barcodes are broadly useful for experimental tasks such as lineage tracing or genetic screens. For such applications, a barcode library would ideally have high diversity (many unique barcodes), nondestructive identification (repeated measurements in the same cells or population), and fast, inexpensive readout (many cells and conditions). Current nucleic acid barcoding methods generate high diversity but require destructive and slow/expensive readout, and current fluorescence barcoding methods are nondestructive, fast, and inexpensive to readout but lack high diversity. We recently proposed a theory for how fluorescent protein combinations may generate a high-diversity barcode library with nondestructive, fast, and inexpensive identification. Here, we present an initial experimental proof-of-concept by generating a library of ∼150 barcodes from two-way combinations of 18 fluorescent proteins, 61 of which are tested experimentally. We use a pooled cloning strategy to generate a barcode library that is validated to contain every possible combination of the 18 fluorescent proteins. Experimental results using single mammalian cells and spectral flow cytometry demonstrate excellent classification performance of individual fluorescent proteins, with the exception of mTFP1, and of most evaluated barcodes, with many true positive rates >99%. The library is compatible with genetic screening for hundreds of genes (or gene pairs) and lineage tracing hundreds of clones. This work lays a foundation for greater diversity libraries (potentially ∼10 and more) generated from hundreds of spectrally resolvable tandem fluorescent protein probes.
基因编码的单细胞条形码广泛应用于谱系追踪或基因筛选等实验任务。对于此类应用，条形码文库理想情况下应具有高多样性（许多独特的条形码）、非破坏性识别（在同一细胞或群体中进行重复测量）以及快速、廉价的读出方式（针对许多细胞和条件）。当前的核酸条形码方法具有高多样性，但需要进行破坏性操作且读出过程缓慢/昂贵，而当前的荧光条形码方法具有非破坏性、读出速度快且成本低的特点，但缺乏高多样性。我们最近提出了一种理论，阐述了荧光蛋白组合如何生成具有非破坏性、快速且廉价识别功能的高多样性条形码文库。在此，我们通过从18种荧光蛋白的双向组合中生成一个约150个条形码的文库，给出了初步的实验概念验证，其中61个条形码经过了实验测试。我们采用混合克隆策略生成了一个条形码文库，经验证该文库包含了18种荧光蛋白的每一种可能组合。使用单个哺乳动物细胞和光谱流式细胞术的实验结果表明，除mTFP1外，大多数评估的条形码中，各个荧光蛋白的分类性能优异，许多真阳性率>99%。该文库与数百个基因（或基因对）的基因筛选以及数百个克隆的谱系追踪兼容。这项工作为从数百种光谱可分辨的串联荧光蛋白探针生成更多样性的文库（可能达到10种及更多）奠定了基础。
