Loveless Theresa B, Carlson Courtney K, Dentzel Helmy Catalina A, Hu Vincent J, Ross Sara K, Demelo Matt C, Murtaza Ali, Liang Guohao, Ficht Michelle, Singhai Arushi, Pajoh-Casco Marcello J, Liu Chang C
Department of Biomedical Engineering, University of California, Irvine, CA, USA.
Center for Synthetic Biology, University of California, Irvine, CA, USA.
Nat Chem Biol. 2025 Apr;21(4):512-521. doi: 10.1038/s41589-024-01764-5. Epub 2024 Nov 14.
Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell's genome, allowing for the later reconstruction of cellular experiences by DNA sequencing. We present a DNA recorder, peCHYRON, that achieves high-information, durable, and temporally resolved multiplexed recording of multiple cellular signals in mammalian cells. In each step of recording, prime editor, a Cas9-reverse transcriptase fusion protein, inserts a variable triplet DNA sequence alongside a constant propagator sequence that deactivates the previous and activates the next step of insertion. Insertions accumulate sequentially in a unidirectional order, editing can continue indefinitely, and high information is achieved by coexpressing a variety of prime editing guide RNAs (pegRNAs), each harboring unique triplet DNA sequences. We demonstrate that the constitutive expression of pegRNA collections generates insertion patterns for the straightforward reconstruction of cell lineage relationships and that the inducible expression of specific pegRNAs results in the accurate recording of exposures to biological stimuli.
基因编码的DNA记录器可将瞬时生物事件无创地转化为细胞基因组中的持久突变，从而能够在之后通过DNA测序重建细胞经历。我们展示了一种DNA记录器peCHYRON，它能够在哺乳动物细胞中对多个细胞信号进行高信息、持久且具有时间分辨率的多重记录。在记录的每个步骤中，引导编辑器（一种Cas9逆转录酶融合蛋白）会在一个恒定的传播序列旁插入一个可变的三联体DNA序列，该传播序列会使上一步失活并激活下一步插入。插入以单向顺序依次累积，编辑可无限期持续，并且通过共表达多种各自携带独特三联体DNA序列的引导编辑向导RNA（pegRNA）实现高信息记录。我们证明，pegRNA集合的组成型表达会生成用于直接重建细胞谱系关系的插入模式，而特定pegRNA的诱导型表达则能准确记录对生物刺激的暴露情况。
